ANTICANCER RESEARCH 33: 1989-2000 (2013)

Evaluation of Melphalan, Oxaliplatin, and Paclitaxel in Colon, Liver, and Gastric Cancer Cell Lines in a Short-term Exposure Model of Chemosaturation Therapy by Percutaneous Hepatic Perfusion

RAJNEESH P. UZGARE, TIMOTHY P. SHEETS and DANIEL S. JOHNSTON

Pharmaceutical Research and Development, Delcath Systems, Inc., Queensbury, NY, U.S.A.

Abstract. Background: The goal of this study was to candidates for use in the CS-PHP system to treat patients determine whether liver, gastric, or colonic cancer may be with gastric and colonic metastases, and primary cancer of suitable targets for chemosaturation therapy with the liver. percutaneous hepatic perfusion (CS-PHP) and to assess the feasibility of utilizing other cytotoxic agents besides Chemotherapeutic molecules exert beneficial clinical effects melphalan in the CS-PHP system. Materials and Methods: by inhibiting cell growth or by inducing cell death via Forty human cell lines were screened against three cytotoxic apoptosis. They can be divided into several categories based chemotherapeutic agents. Specifically, the dose-dependent on their mechanisms of action. The chemotherapeutic agent effect of melphalan, oxaliplatin, and paclitaxel on melphalan hydrochloride, which has been approved by the proliferation and apoptosis in each cell line was evaluated. US Food and Drug Administration and is used in the These agents were also evaluated for their ability to induce treatment of multiple myeloma and ovarian cancer, is a apoptosis in normal primary human hepatocytes. A high- derivative of nitrogen mustard that acts as a bifunctional dose short-term drug exposure protocol was employed to alkylating agent. Melphalan causes the alkylation of DNA at simulate conditions encountered during CS-PHP. Results: the N-7 position of guanine and the N-3 position of adenine The average concentration of melphalan required for (1). Thus, the binding of melphalan to DNA can result in inducing significant apoptosis was 61 μM, or about 3-fold cross-linking between bases, particularly G-G and G-A, on less than the theoretical concentration of 192 μM, achieved complementary strands, which leads to double-stranded DNA in the hepatic artery during CS-PHP dosing with melphalan. breaks and cell death through a caspase-mediated apoptotic Additionally, we found that gastric cancer cell lines were 2- pathway (2-5). Taxanes, such as paclitaxel, exert their effects 5 fold more sensitive to apoptosis than liver cancer cell lines by inhibiting microtubules (6, 7). Disruption of microtubules to all three compounds, suggesting that in addition to colonic during cell division leads to cell-cycle arrest and subsequent and gastric cancer metastases to the liver, primary gastric cell death by apoptosis. Paclitaxel has been used clinically cancer may also be amenable to management by CS-PHP in treatment regimens for a number of cancer types such as using an appropriate therapeutic agent. Significantly, at breast cancer and non–small-cell lung cancer (8). Platinum concentrations that are predicted using the CS-PHP system, agents, such as oxaliplatin, bind to and damage DNA, which these agents caused apoptosis of colonic, gastric, and liver leads to disruption of transcription and replication and to cancer cells but were not toxic to primary human cell-cycle arrest. If this DNA damage is not repaired, hepatocytes. Conclusion: The compounds tested are potential apoptosis will ultimately result (9). These agents comprise part of the treatment regimens for cancer such as colorectal, ovarian, and bladder cancer (10). At the doses required for clinical efficacy, chemotherapeutic Correspondence to: Rajneesh P. Uzgare, Ph.D., Director of agents are often associated with significant side effects. For Pharmaceutical Research, Delcath Systems, Inc., 566 Queensbury example, because these compounds target rapidly dividing Avenue, Queensbury, NY 12804, U.S.A. Tel: +1 5187438892 ext. 349, Fax: +1 5187430139, e-mail: [email protected] cells, they often induce myelosuppression, which contributes significantly to patient morbidity (11). Thus, a system that Key Words: Chemosaturation therapy, percutaneous hepatic enables the delivery of chemotherapeutic compounds perfusion, melphalan, taxanes, platinum agents. specifically to the organ of interest while reducing systemic

0250-7005/2013 $2.00+.40 1989 ANTICANCER RESEARCH 33: 1989-2000 (2013) exposure offers significant advantages. Isolated hepatic cytotoxicity assays, cell lines were grown in Roswell Park Memorial perfusion (IHP) and percutaneous hepatic perfusion (PHP) Institute 1640 medium (RPMI 1640) or Dulbecco’s modified offer this type of delivery system. Eagle’s medium (DMEM), 10% fetal bovine serum (FBS), 2 mM L-alanyl-L-glutamine, and 1 mM sodium pyruvate in a humidified The administration of melphalan via IHP and PHP has atmosphere of 5% CO at 37˚C. Cells were seeded into 384-well been studied for the treatment of liver cancer, as well as for 2 plates and incubated in 5% CO2 at 37˚C. metastases to the liver from colorectal cancer and metastatic Human primary hepatocytes (Invitrogen, Carlsbad, CA, USA) melanoma, among others (12-15). IHP permits intensification were thawed in hepatocyte culture medium with supplements and of chemotherapy to the liver, thereby improving efficacy growth factors [hepatocyte basal medium (HBM™) and hepatocyte while limiting extrahepatic toxicity. However, its efficacy is culture medium (HCM™) SingleQuots® from LONZA, restricted because the treatment cannot be repeated since Walkersville, MD, USA], treated with a Percoll (Sigma-Aldrich, St. Louis, MO, USA) separation step, and immediately seeded into 384- post-surgical intra-abdominal adhesions preclude further well collagen I-coated optical plates (BD Biosciences, San Jose, surgery. By contrast, chemosaturation therapy with PHP (CS- CA, USA). Cells were incubated at 37˚C in 5% CO2 for 24 h, after PHP), represented in Figure 1, is a repeatable percutaneous which the medium was exchanged and the cells were treated with procedure for the intra-arterial administration of melphalan test compounds. hydrochloride to the liver with extracorporeal filtration. The CS-PHP device is approved for clinical use in Europe where Cell treatment with test compounds. Melphalan, oxaliplatin, cases are being performed routinely in several countries. The paclitaxel and mitoxantrone were purchased from Sigma-Aldrich or proprietary CS-PHP system delivers melphalan to the liver Calbiochem (EMD Millipore, Billerica, MA, USA). For the via the proper hepatic artery and employs a double-balloon multiplexed cytotoxicity assays, compounds were serially diluted 3.16-fold and assayed over a range of 10 concentrations; the highest aspiration catheter in the retrohepatic inferior vena cava to and lowest concentration for each compound is specified in Tables isolate and collect hepatic venous outflow and direct the I-III. The final dimethyl sulfoxide (DMSO) concentration in the effluent to an extracorporeal circuit containing a proprietary assay did not exceed 0.1%. Compounds were added 24 h after cell hemofiltration system that reduces the concentration of seeding. At the same time, a time-zero untreated cell plate was melphalan in the blood dramatically and returns the filtered generated. The compound treatment period was 50 min. After blood to the systemic circulation through the jugular vein. incubation with a compound, the cells were spun, the supernatant Thus, when combined with an appropriate filtration system, was aspirated to remove the compound, and the medium was exchanged three times and cells incubated for an additional 72 h at this system could potentially be used to deliver other 37˚C. chemotherapeutic agents specifically to the liver. For primary hepatocytes, at 24 h after plating, the medium was In this study, we screened six gastric, seven liver, and 27 exchanged and compounds were added. Each plate of tested colon cancer cell lines for their sensitivity to melphalan, compounds included one reference compound (mitoxantrone) and oxaliplatin, and paclitaxel. We determined whether any of vehicle control. Compounds were serially diluted 3.16-fold and these agents effectively induces cell death of cancer cells in assayed over a range of 10 concentrations (0.008 μM - 250 μM for vitro and, thus, could be used with the CS-PHP device. This mitoxantrone) similar to those described for the cell lines. The final DMSO concentration in the assay did not exceed 0.5%. The compound study is notably relevant to clinical settings because of the treatment period was 1 or 4 h, followed by a wash-out. Fresh medium short time of exposure (50 min) of cells to high doses of the was added to the cells, and the cells were incubated for an additional compound of interest followed by a wash-out step to remove 17 or 14 h, respectively, at 37˚C prior to being assayed for an attached the agent from the cell milieu, simulating CS-PHP. A live count and for caspase-3 activation at 18 h. multiplex system was used to simultaneously measure cell proliferation and apoptosis. Finally, in order to address the Cell fixation and labeling. Following the final incubation step, the safety of these agents in a potential CS-PHP to treat liver cells were fixed and stained with fluorescently labeled antibodies metastases, we performed assays on primary human and nuclear dye to allow visualization of nuclei and apoptotic cells. hepatocytes and measured the relative attached cell count and Cells were fixed with paraformaldehyde (3.33% final concentration). Plates were spun briefly and incubated for 15 min apoptosis induction. at room temperature. The supernatant was aspirated using a BioTek Elx405 plate washer (BioTek, Winooski, VT, USA) and 80 μl of Materials and Methods block solution was added [0.25% bovine serum albumin (BSA), 0.05% Triton X-100, and 0.25% goat serum in tris-buffered saline Cell lines and human hepatocytes. The 40 cell lines used in this (TBS) buffer] for 10 min. A rabbit polyclonal anti-active caspase-3 study and their tissue of origin are shown in Table I. All cell lines (Cell Signaling Technology, Danvers, MA, USA) was added to the were obtained from the American Type Culture Collection plates after aspiration of the block solution and cells then incubated (Manassas, VA, USA), Leibniz-Institut Deutsche Sammlung von for 30 min. Plates were then washed twice with PBS and secondary Mikroorganismen und Zellkulturen GmbH (Braunschweig, antibody was added for 30 min (Alexa Fluor 488 goat anti-rabbit, Germany), and the Japanese Collection of Research Bioresources Invitrogen). The plates were then washed twice, stained with 2 (National Institute of Health Sciences, Osaka, Japan) and mg/ml amidino phenyl indole (DAPI; Invitrogen) and imaged using maintained as specified by the vendor. For the multiplexed a General Electric (GE) Healthcare (Waukesha, WI, USA) IN Cell

1990 Uzgare et al: MPL, OXA, and PAX Chemosaturation Therapy by CS-PHP

Table I. Summary of melphalan sensitivity. Cell lines are ranked from Table II. Summary of results for oxaliplatin sensitivity. Cell lines are sensitive to resistant to the induction of 5-fold apoptosis. The tissue of ranked from sensitive to resistant to the induction of 5-fold apoptosis. origin of each cell line is indicated as gastric (G), liver (L) or colonic The tissue of origin of each cell line is indicated as gastric (G), liver (C). (L) or colonic (C).

Melphalan† Oxaliplatin†

Conc. for 5-fold EC50 (μM) Conc. for 5-fold EC50 (μM) apoptosis induction (μM) apoptosis induction (μM)

Cell line Cell line HCT-8 (C) 2.89 2.54 Colo 320 HSR (C) 15.50 15.70 RKO-AS45-1 (C) 4.24 4.25 HCT-116 (C) 19.80 12.40 Colo 320 HSR (C) 4.83 2.92 SNU-1 (G) 20.70 20.50 HCT-15 (C) 5.05 7.17 AGS (G) 25.90 4.71 SW620 (C) 5.14 6.69 LS-174T (C) 28.70 7.83 RKOE6 (C) 6.03 8.07 SW48 (C) 31.40 11.60 RKO (C) 7.29 7.54 HCT-15 (C) 52.30 40.80 DLD-1 (C) 7.95 12.70 DLD-1 (C) 52.60 51.70 Colo 205 (C) 9.68 0.68 Colo 205 (C) 56.30 46.20 SW48 (C) 10.50 6.33 SNU-5 (G) 66.90 49.20 HLF (L) 12.00 9.43 OCUG-1 (L) 68.90 466.00 HCT-116 (C) 12.40 14.00 NCI-H508 (C) 73.30 7.64 AGS (G) 14.70 4.29 HUH-6 Clone 5 (L) 80.70 17.20 KATO III (G) 15.40 13.20 HepG2 (L) 91.80 35.60 SNU-16 (G) 16.80 30.00 KATO III (G) 92.90 6.37 SNU-1 (G) 18.00 8.92 Colo 320DM (C) 97.20 44.20 NCI-H508 (C) 18.10 26.60 RKO (C) 98.70 67.50 LS-174T (C) 19.80 1.24 SW620 (C) 118.00 23.30 HT-29 (C) 23.90 25.50 SNU-16 (G) 120.00 28.60 HLE (L) 26.20 18.80 RKOE6 (C) 130.00 52.90 WiDr (C) 28.60 17.50 SW837 (C) 134.00 11.20 SW480 (C) 32.20 54.60 SW1463 (C) 157.00 46.50 SW403 (C) 32.70 22.20 HLF (L) 199.00 210.00 OCUG-1 (L) 40.00 72.40 HLE (L) 214.00 101.00 SW837 (C) 46.00 11.20 HT-29 (C) 237.00 36.60 HuCCT1 (L) 46.40 26.20 SW480 (C) 270.00 41.90 Colo 320DM (C) 50.30 5.13 LS1034 (C) 336.00 15.90 SNU-5 (G) 59.60 167.00 SW1116 (C) 446.00 14.40 LS1034 (C) 67.30 26.10 HCT-8 (C) N/A 28.20 HUH-6 Clone 5 (L) 89.00 13.00 RKO-AS45-1 (C) N/A 57.60 HepG2 (L) 92.30 14.60 WiDr (C) N/A 27.50 SW1463 (C) 100.00 56.90 SW403 (C) N/A 62.20 T84 (C) 138.00 29.70 HuCCT1 (L) N/A 77.30 SW1417 (C) 183.00 77.00 T84 (C) N/A 21.80 Colo 201 (C) 278.00 39.30 SW1417 (C) N/A 65.90 NCI-H747 (C) 279.00 22.30 Colo 201 (C) N/A 60.70 SW1116 (C) 450.00 23.30 NCI-H747 (C) N/A 308.00 HS 746T (G) N/A 61.60 HS 746T (G) N/A 26.50 SNU-423 (L) N/A 27.70 SNU-423 (L) N/A N/A SW948 (C) N/A 24.60 SW948 (C) N/A 30.00

†Range tested: 0.01 μM - 500 μM. †Range tested: 0.01 μM - 500 μM.

Analyzer 1000. Twelve-bit TIFF images were acquired using the IN by the signal intensity of the incorporated nuclear dye. The cell Cell Analyzer 1000 3.2 and analyzed using Developer Toolbox 1.6 proliferation assay output is the relative cell count. To determine the software (GE Healthcare, Piscataway, NJ, USA). cell proliferation endpoint of EC50, data output was transformed to a percentage that of the control (POC) using the following formula: Cell proliferation assessment (EC50). Cell proliferation was assessed using a cell image-based technique. Cells were stained with DAPI, POC=relative cell count (compound wells) ÷ relative cell count a nuclear dye, to visualize nuclei. Cell proliferation was measured (vehicle wells) ×100

1991 ANTICANCER RESEARCH 33: 1989-2000 (2013)

Table III. Summary of results for paclitaxel sensitivity. Cell lines are Table IV. Apoptosis induction by tissue of origin. ranked from sensitive to resistant to the induction of 5-fold apoptosis. The tissue of origin of each cell line is indicated as gastric (G), liver Avg. conc. (μM) for apoptosis induction (L) or colonic (C). Cell line: Tissue of origin Melphalan Oxaliplatin Paclitaxel Paclitaxel† All 60.9 130.4 3.5 Conc. for 5-fold apoptosis EC50 (μM) Liver 51.0 183.4 0.9 induction (μM) Colon 70.1 130.8 4.8 Gastric 24.9 65.3 0.2 Cell line OCUG-1 (L) 0.0130 0.1820 SNU-1 (G) 0.0180 0.0141 HCT-116 (C) 0.0328 0.0265 RKOE6 (C) 0.0480 0.0495 RKO-AS45-1 (C) 0.0669 0.0308 SNU-16 (G) 0.0683 0.0233 Apoptosis assay. Apoptotic cells were detected using an antibody to HuCCT1 (L) 0.0994 0.0549 anti-activated caspase-3 (rabbit polyclonal anti-active caspase-3; NCI-H508 (C) 0.1070 0.0626 Cell Signaling Technology). Automated fluorescence microscopy RKO (C) 0.1120 0.0532 was carried out using a GE Healthcare IN Cell Analyzer 1000, and LS-174T (C) 0.1370 0.0736 images were collected with a 4 × objective. HT-29 (C) 0.1750 0.1530 For apoptosis output (indicated by activated caspase-3 marker), Colo 205 (C) 0.2520 0.1170 each intensity was first normalized to the nuclear intensity in their KATO III (G) 0.2820 0.0565 respective wells. This ratio was then normalized to that of the AGS (G) 0.3360 0.1560 control wells and expressed as fold induction over vehicle: WiDr (C) 0.3550 0.2380 Apoptosis fold induction= (Cx/Nx) ÷ (Cc/Nc) HepG2 (L) 0.4110 0.5370 where Cx=caspase intensity for well X, Nx=nuclear intensity for SW620 (C) 0.4200 0.2250 well X, Cc=caspase intensity for control well, and Nc=nuclear SW403 (C) 0.4380 0.1800 intensity for control well. Wells with concentrations higher than the HUH-6 Clone 5 (L) 0.4440 0.3480 relative cell count (IC95) were eliminated from the caspase-3 SW1417 (C) 0.5680 0.1560 induction analysis. SW480 (C) 0.5820 0.1980 The output is reported as the fold increase in apoptotic cells over HCT-8 (C) 1.0700 1.3400 vehicle background normalized to the relative cell count for each SW837 (C) 1.6600 1.1600 well. Significant induction of apoptosis was defined based on HLF (L) 1.9300 1.7400 vehicle mean plus six standard deviations (SDs). When the vehicle HLE (L) 2.8000 1.2300 mean plus six SDs was compared for multiple cell lines, the overall DLD-1 (C) 3.6900 3.7200 range of apoptosis induction, as measured by activation of caspase- Colo 320 HSR (C) 3.9100 8.5500 3, was 2.5-fold to 4.5-fold. Hence, 5-fold apoptosis induction was NCI-H747 (C) 8.8500 0.0551 selected as being significant, and concentrations of each agent that HCT-15 (C) 10.7000 11.9000 result in 5-fold apoptosis induction are reported. LS1034 (C) 16.6000 7.5100 SW1463 (C) 26.1000 2.5300 Primary hepatocyte cytotoxicity assay: Staining. For primary Colo 320DM (C) 28.9000 25.9000 hepatocytes, following the 18-h incubation period, the cells were SW48 (C) N/A 0.1920 incubated for 30 min at 37˚C with multiplexed fluorescent dyes and SNU-5 (G) N/A 0.2750 SW1116 (C) N/A 57.1000 then imaged. Nuclei were labeled with DAPI for cell quantification. T84 (C) N/A 10.8000 The cells were then fixed with 3% paraformaldehyde and imaged. Colo 201 (C) N/A 0.0584 Automated fluorescence microscopy was carried out using a GE HS 746T (G) N/A 0.3470 Healthcare IN Cell Analyzer 1000, and images were collected with SNU-423 (L) N/A 2.8900 a 4 × objective. SW948 (C) N/A 0.5500 Attached Live Cell Count: For primary human hepatocytes, live cell attachment was measured by the degree of nuclear segmentation †Range tested: 0.003 μM - 100 μM. of the incorporated nuclear dye. The live cell attachment assay output is referred to as the percentage of attached live cells. To determine the live cell attachment end point, the nuclear segmentation data output was transformed to POC using the following formula: Relative cell count (EC50) is the test compound concentration at the POC=[nuclear segmentation (compound wells)] ÷ [nuclear curve inflection point or half the effective response. EC50 values segmentation (vehicle wells)] ×100 were calculated using nonlinear regression analysis to fit the data to The EC50 for attached live cells is the concentration of test a sigmoidal 4-point, 4-parameter, one-site dose response model, compound that produces 50% of the maximum effective response at where y (fit)=A + [(B – A) ÷ (1 + [C/x]D)]. the curve inflection point.

1992 Uzgare et al: MPL, OXA, and PAX Chemosaturation Therapy by CS-PHP

Figure 1. The chemosaturation therapy with percutaneous hepatic perfusion (CS-PHP) system. The CS-PHP system enables the isolation of the blood supply to the liver through the use of a catheter system, thus allowing, via repeated procedures over time, the direct delivery of chemotherapeutic agents to the liver. The blood containing free chemotherapeutic agent is captured between the two balloons in the isolation aspiration catheter and is directed to an extracorporeal circuit containing a proprietary filter that filters the chemotherapeutic agent from the blood, significantly reducing the concentration of the chemotherapeutic agent in the blood before it is returned to the systemic circulation through the jugular vein. This allows the patient to be treated with relatively high doses of chemotherapy agents over a 60-minute period while limiting extrahepatic exposure to melphalan, thereby greatly reducing systemic toxicity. The figure shown is the property of Delcath Systems, Inc.

Results colorectal adenocarcinoma cell lines Colo 205 and LS-174T were the most sensitive, and the colorectal adenocarcinoma Cancer cell line sensitivity to melphalan. The 40 cell lines that cell line SW1417 and the gastric carcinoma cell line SNU-5 were tested in this study had a range of sensitivities to were the most resistant. melphalan, defined by the induction of apoptosis and EC50 (Table I). The concentration required for 5-fold induction of Cancer cell line sensitivity to oxaliplatin. As shown in Table apoptosis ranged from 2.89 μM to 450 μM. The cancer cell II, 5-fold induction of apoptosis was achieved at oxaliplatin lines that were most sensitive to melphalan-induced apoptosis concentrations ranging from 15.5 μM to 446 μM. Colo 320 were the HCT-8 colorectal adenocarcinoma and the RKO- HSR and HCT-116 colorectal carcinoma cells exhibited the AS45-1 colon carcinoma cell lines. Among the cell lines in greatest sensitivity to oxalipatin. Among the cell lines for which apoptosis could be quantified, the most resistant were which apoptosis could be quantified, the most resistant were the colorectal adenocarcinoma lines NCI-H747 and the the colorectal adenocarcinoma lines LS1034 and SW1116. SW1116. In three cell lines, namely the HS 746T gastric Maximum apoptosis induction was less than 5-fold in 12 cell carcinoma, SNU-423 hepatocellular carcinoma, and SW948 lines. The range of EC50 values was 4.71 μM to 466 μM. colorectal adenocarcinoma, 5-fold induction of apoptosis was The most sensitive cell lines were AGS and KATO III, both not achieved at any of the concentrations tested. The range for of which are gastric carcinomas. Among the cell lines with EC50 was 0.68 to 167 μM. When assessed by EC50, the measurable EC50 values, the colorectal carcinoma NCI-H747

1993 ANTICANCER RESEARCH 33: 1989-2000 (2013)

Figure 2. Concentration required for a 5-fold induction of apoptosis in all cell lines for all compounds tested. Note that for cell lines for which 5- fold induction of apoptosis was not achieved, the value used to construct the graph was the highest concentration of that agent tested.

and gall bladder carcinoma OCUG-1 cell lines were highly apoptosis were the colorectal adenocarcinoma lines SW1463 resistant to oxaliplatin. The most resistant cell line was SNU- and Colo 320 DM. The cells most sensitive to paclitaxel, as 423, a hepatocellular carcinoma, for which an EC50 could determined by EC50, were SNU-16 and SNU-1 gastric not be determined. carcinoma cells. The colorectal adenocarcinoma cell lines Colo 320 DM and SW1116 were the least sensitive. Cancer cell line sensitivity to paclitaxel. Quantifiable apoptosis was detected in 32 out of the 40 cell lines (Table III). Comparison of sensitivity of cancer cell Lines to all agents Concentrations required for 5-fold induction of apoptosis were tested. Shown in Figures 2 and 3 are radar graphs of the 0.013 μM to 28.9 μM. The cells requiring the lowest sensitivity of each cell line to the three compounds tested in concentration of paclitaxel for 5-fold induction of apoptosis this study. Paclitaxel was the most potent of the three drugs, were OCUG-1 gall bladder carcinoma cells and SNU-1 gastric with apoptosis induction and EC50 concentration ranges in the carcinoma cells. The cells most resistant to paclitaxel-induced double-digit nanomolar range for sensitive cell lines. For

1994 Uzgare et al: MPL, OXA, and PAX Chemosaturation Therapy by CS-PHP

Figure 3. Relative cell count. EC50 in all cell lines, for all compounds tested.

melphalan and oxaliplatin, these concentrations were found to In fact, in the case of SNU-423 cells, an EC50 value could not be several orders of magnitude higher and were calculated to be calculated for oxaliplatin. In the oxaliplatin-treated group, be in the single- and double-digit micromolar range, there were 12 cell lines (the most among the three drugs) for respectively. Among the cell lines in which no measurable which 5-fold apoptosis was not detected. The paclitaxel- and drug-induced apoptosis was detected, 10 (HCT-8, RKO-AS- melphalan-treated groups had eight and three cell lines, 45-1, SW48, WiDr, SW403, HuCCT1, SNU-5, SW1417, NCI- respectively, in which 5-fold apoptosis was not detected. H747, and SW1116) were resistant to one drug, two (T84 and The 40-cell–line panel used in this study consists of cell Colo 201) were resistant to two drugs, and three (Hs746T, lines derived from gastric (n=6), liver (n=7), and colon (n=27) SNU-423, and SW948) were resistant to all three drugs. When cancer. Table IV the average concentration of each drug compared with the other cell lines used in this study, the three required to induce apoptosis according to tissue of origin. The cell lines resistant to all three drugs were also relatively average concentration of melphalan required to induce resistant when assayed for the antiproliferative EC50 endpoint. apoptosis across all tissue types tested was 60.9 μM. For the

1995 ANTICANCER RESEARCH 33: 1989-2000 (2013)

Figure 4. Human hepatocyte cytotoxicity and apoptosis upon exposure to high-dose compound for 1 and 4 hours: (A) melphalan, (B) oxaliplatin, (C) paclitaxel, and (D) mitoxantrone.

liver-specific subset, this dropped to 51 μM, whereas for colon 1 h then washed off the drug, replenished the cells with a cancer cell lines, it was 70.1 μM. The average melphalan fresh drug-free medium, and carried out assays for apoptosis concentration required for apoptosis induction in the gastric and cytotoxicity at 18 h. We also assessed the potential cell lines was more than 2-fold lower at 24.9 μM,with the safety window of CS-PHP by simulating conditions in which concentration being in the range of 14 to 18 μM for four (AGS, hepatocytes were exposed to high doses of drug for 4 h. KATO III, SNU-16, and SNU-1) of the five gastric cancer cell The IC50 was used as a measure of cell detachment or lines (Table I). A similar trend was observed for oxaliplatin and cytotoxicity and caspase-3 activation as a measure of apoptosis. paclitaxel, where the average concentrations required for the The results of these assays are shown in Figure 4. None of the induction of apoptosis in gastric cancer cell lines were lower three compounds at any of the concentrations or time points than those required for liver and colonic cancer cell lines. tested showed any measurable cytotoxicity or caspase-3 activation. However, the reference control agent, mitoxantrone, Hepatic toxicity assay using human primary hepatocytes. A was found to be cytotoxic at 1 and 4 h, with an EC50 of multiplexed hepatic toxicity assay was performed to evaluate approximately 8 μM. Mitoxantrone also induced measurable the effect of short-term exposure of the three agents on apoptosis at the second highest concentration, 79 μM. The primary human hepatocytes. The hepatic CS-PHP procedure highest concentration of mitoxantrone, 250 μM, was found to performed in the clinic consists of a 30-min drug infusion be severely cytotoxic (<5% of the cells were viable) and, thus, period followed by a 30-min wash-out period. In order to accurate measurements of apoptosis were not possible. It was simulate these conditions, we exposed the cells to drugs for also determined that melphalan, oxaliplatin, and paclitaxel, at

1996 Uzgare et al: MPL, OXA, and PAX Chemosaturation Therapy by CS-PHP the concentrations used in this study, do not cause affecting the liver would result in a theoretical concentration in mitochondrial damage or the induction of reactive oxygen the range of 94 to 135 μM, which is several orders of magnitude species (data not shown). Taken together, these data suggest greater than the average of 3.5 μM (Table IV) required to kill the that melphalan, oxaliplatin, and paclitaxel, at the concentrations majority of the cancer cell lines in our study. The MTD for i.v. and time employed in these studies, are not toxic to primary oxaliplatin was determined to be 135 mg/m2 in a dose escalation human hepatocytes and may be well tolerated by the liver. phase I clinical trial in patients with advanced cancer (19). A subsequent phase II study with oxaliplatin as a single agent at Discussion 130 mg/m2 to treat patients with colorectal carcinoma patients reported modest response rates of 20% (20). Intrahepatic delivery In this study, we used a high-dose, short-term drug exposure of oxaliplatin via CS-PHP at 130 mg/m2 would achieve a protocol to measure the chemosensitivity of six gastric, seven theoretical concentration of 151 μM, which is almost identical liver, and 27 colon cancer cell lines to three commonly used to the average concentration of 130 μM required to induce chemotherapeutic agents, namely melphalan, oxaliplatin, and apoptosis in our study. Interestingly, neither of these two studies paclitaxel. Specifically, we tested the ability of these drugs reported any hepatotoxicity in patients who were given i.v. to induce apoptosis and inhibit cell proliferation. We also oxaliplatin at 130 mg/m2, which indicates that even higher doses tested the ability of any of these agents to cause apoptosis might be tolerated by the liver if the drug were delivered and/or cytotoxicity in primary human hepatocytes at high specifically to the liver via CS-PHP. Indeed, a more recent study doses. The treatment protocol was designed to assess the demonstrated that the MTD of oxaliplatin delivered via hepatic feasibility of extending the applicability of Delcath’s arterial infusion was 140 mg/m2, and this dose, when used in proprietary CS-PHP system by simulating conditions combination with other systemic therapies had antitumor activity encountered during the clinical procedure. Given the high in patients with metastatic liver disease (21). These data indicate doses of agents employed over a short time during CS-PHP, that oxaliplatin delivered via CS-PHP may be effective in induction of apoptosis is likely to be the key determinant of treating patients with liver metastasis. cell death. The most common origin of metastases confined The effect of melphalan, oxaliplatin, and paclitaxel on tumor to the liver is colorectal cancer (12), making colonic, gastric, cell killing is all the more striking when viewed in light of the and liver cancer cells logical targets for the CS-PHP system. fact that none of these drugs had a deleterious effect on In this study, the average melphalan concentration of 60.9 μM hepatocyte viability or toxicity, as assessed in our study. This is required for induction of 5-fold apoptosis in cells from gastric, an important consideration, because high doses of drug can be colonic, and liver cancer was lower than the theoretical repeatedly administered via CS-PHP to achieve tumor ablation. concentration achieved in the hepatic artery at the site of drug The maintenance of hepatocyte cell viability and a lack of infusion during CS-PHP. In a phase I dose escalation trial in apoptosis induction even after extended exposure periods of one patients with unresectable hepatic malignancies, the maximum and four hours clearly demonstrate that these drugs are well tolerated dose (MTD) for melphalan delivered via CS-PHP was tolerated by hepatocytes. There are several lines of clinical and in determined to be 3 mg/kg (15). In a subsequent phase III trial vitro evidence that support our data. Firstly, although systemic using melphalan at 3 mg/kg, up to a maximum dose of 220 mg, cytoreductive therapy using i.v. high-dose melphalan at 100 in 92 patients with hepatic metastases from malignant mg/m2 has been shown to result in hepatotoxicity, manifested as melanoma, the investigators reported a hepatic progression-free veno-occlusive disease (22), the aforementioned phase I clinical survival (H-PFS) of 245 days, which was significantly longer trials using high doses of melphalan via CS-PHP found no than the H-PFS of 49 days seen in the control group (16). hepatotoxicity associated with doses below the MTD but at Assuming that approximately one fourth of the total hepatic which antitumor effects were observed (15). Secondly, circulation comes from the hepatic artery, a 220-mg dose of melphalan induced caspase-3-dependent apoptosis of murine melphalan infused over 30 min at an extracorporeal circuit flow hepatocytes in vitro was shown to occur in the range of 100 to rate of 500 ml/min would result in a theoretical concentration of 200 μg/ml (327-654 μM) but required an overall exposure time 192 μM (220 mg in 3.75 l of blood) in the hepatic artery. This of 9 to 12 h (5). Moreover, there was no increase in alanine represents a 3-fold increase over the average concentration aminotransferase (ALT), a marker of liver health and function, required to induce apoptosis in the cell lines used in our study. A in mouse livers perfused with 150 mg/kg of melphalan until 360 similar argument can be made for the use of paclitaxel through min (5). Intrahepatic delivery of melphalan via CS-PHP results CS-PHP to treat patients with metastases to the liver. Clinical in a far lower theoretical concentration (192 μM) over a much trials with intravenous (i.v.) paclitaxel to treat patients with breast shorter time period (30 min) and, thus, would be predicted not to cancer have used doses ranging from 175 mg/m2 (17) to 250 result in significant apoptosis of hepatocytes. Thirdly, oxaliplatin mg/m2 (18), which for an average individual with a body surface at i.v. doses of 130 mg/m2 was shown to be equally well area of 1.73 m2 translates to 303 to 433 mg. Intrahepatic delivery tolerated by patients regardless of hepatic function, which of paclitaxel at this dose via CS-PHP to treat patients with cancer suggests that it does not result in hepatotoxicity (23). Fourthly,

1997 ANTICANCER RESEARCH 33: 1989-2000 (2013) clinical studies with 250 mg/m2 i.v. paclitaxel given as a 3-h exposure. In our study, we plated cells in the log phase and infusion to treat patients with breast cancer did not show any allowed them to adhere overnight before drug exposure. This evidence of drug-related hepatotoxicity, which indicates that it ensured that the added drug encountered rapidly dividing cells is safe and well tolerated by the liver (17). Hepatotoxicity to in their most sensitive state for drug efficacy. It is likely that in paclitaxel has been reported in patients with impaired hepatic the absence of a rapidly dividing cell population in their function (24). Consequently, any use of paclitaxel via CS-PHP in experimental set up, Boulin et al. (26) and van Iersel et al. (27) these patients needs to be considered carefully. Finally, even would have failed to see effects at the lower concentrations. though our primary hepatocyte cytotoxicity assays showed that Moreover, neither of these studies investigated apoptosis as an the three compounds tested are not cytotoxic towards these cells, endpoint in their study, whereas apoptosis, according to us, it is important to note the limitations of this study: hepatocytes represents an important component of the cell death process. represent only one cell type in a complex organ. The liver This is especially relevant in CS-PHP, where tumor cell killing contains many other cell types, such as Kupffer cells, which may due to short-term exposure to a high dose of a therapeutic agent respond differently to these agents. Because hepatocytes are would most likely be a result of apoptosis. nondividing cells, and cells such as Kupffer cells are dividing An unexpected and interesting finding of our study is that cells, they may be more sensitive to the agents tested. gastric cancer cell lines were 2- to 4-fold more sensitive than The potential use of chemotherapeutic drugs in hepatic arterial liver and colonic cancer cell lines across all three drugs. This infusion has been investigated in several studies using ex vivo greater sensitivity of gastric cancer cell lines to melphalan, and in vitro cell-based assays (25-27). A 2-h exposure to oxaliplatin, and paclitaxel is indicative of a drug-specific oxaliplatin was found to result in the dose-dependent inhibition response and not a general increase in sensitivity to all drugs, of proliferation of the HT-29 colon cancer cell line as well as because for certain other drugs the average concentration nine out of 10 tumors derived from colorectal and pancreatic required to induce apoptosis in gastric cancer cell lines is equal cancer metastasis to the liver (25). The average IC50 for the to or greater than that required for apoptosis to be induced in inhibition of colony formation of the metastatic cells was liver cancer cell lines (data not shown). To our knowledge, this reported to be less than 10 μg/ml (25), which corresponds to 25 is the first demonstration that melphalan is more effective μM and is in general agreement with our average EC50 values against cancer cell lines of gastric origin than those of liver of 23 and 45 μM for gastric and colonic cancer cell lines, origin when applied in a simulated CS-PHP dosing regimen. respectively (as calculated from the values for the respective cell This has important implications, not just for treating patients lines shown in Table II). Two other recent studies have also with gastric cancer metastases to the liver, but also for treating investigated in vitro dosing paradigms similar to our study (26, patients with primary gastric cancer via techniques similar to 27). Boulin et al. tested 11 chemotherapeutic drugs, including CS-PHP. Indeed, intraperitoneal chemotherapy using abdominal oxaliplatin and paclitaxel, on three human hepatocellular perfusion for gastric carcinomas has been reported for carcinoma cell lines to select a candidate for transarterial mitomycin C, both as a single agent (28) and in combination chemoembolization (26). The only cell line common to our study with 5-fluorouracil and cisplatin (29). Given our current data, it and theirs was HepG2. However, they report substantially higher would be interesting to speculate that melphalan, oxaliplatin, IC50 values for oxaliplatin when compared to our study (262 μM and paclitaxel could be used via chemosaturation to treat vs. 36 μM) and paclitaxel (241 μM vs. 0.5 μM.) Moreover, in primary gastric cancer. Such evaluations would be predicated their study, there does not appear to be a difference in the IC50 on these drugs not being cytotoxic to cells in the normal gastric for oxaliplatin and paclitaxel, whereas we found a clear epithelium, similar to what we have demonstrated in this study difference (36 μM vs. 0.5 μM). In another study, investigators for normal human hepatocytes. used an in vitro cell line screen and evaluated the potential use of Recent advances in molecular medicine have led to a greater oxaliplatin in combination with melphalan for the treatment of understanding of the genetic heterogeneity of cancer. patients with colorectal cancer to identify dosing schedules that Personalized medicine allows for cancer therapies to be could be employed during isolated liver perfusion (27). Five cell developed for and employed in a specific set of patients who lines- SW48, RKO, DLD-1, HT-29, T84, and SW480 -in their are stratified based on the expression of certain biomarkers. panel of 13 lines were also tested by us in this study. Again, a Notable examples of such approved targeted therapeutics ® majority of the IC50 values reported by them are significantly include the monoclonal antibody trastuzumab (Herceptin ; higher than the EC50 values identified in our study, although it is Roche, Madison, WI, USA), which is used for the treatment of important to point out that the rank order of sensitivity of these patients with human epidermal growth factor receptor 2 (HER- lines to each drug is similar to what we found. One possibly 2)–positive metastatic breast cancer; the small-molecule important reason for the differences seen in our study versus both inhibitor imatinib (Gleevec®; Novartis, Carlsbad, CA, USA), of the recent studies could be the state of plated cells prior to which is used to treat patients with chronic myelogenous drug exposure. In both of these studies, cells were plated and leukemia harboring the breakpoint cluster region-Abelson allowed to reach confluence for three to four days prior to drug leukemia (BCR-ABL) translocation and gastrointestinal stromal

1998 Uzgare et al: MPL, OXA, and PAX Chemosaturation Therapy by CS-PHP tumors carrying a tyrosine protein kinase Kit (c-KIT) mutation be applied at effective concentrations using the CS-PHP (30-32). A need to identify biomarkers predictive of system, without inducing liver toxicity, to treat primary and chemotherapy response has led to large screenings of multiple metastatic liver cancer. human cancer cell lines against multiple drugs (33). Previous work in this area has generally focused on single-gene References mutations, but some studies have also analyzed the association of gene expression profiles with the response of different cell 1 Souliotis VL, Dimopoulos MA and Sfikakis PP: Gene-specific formation and repair of DNA monoadducts and interstrand cross- lines to individual agents. Oncogenes whose products are direct links after therapeutic exposure to nitrogen mustards. Clin targets of relevant drugs and inactivating mutations of tumor- Cancer Res 9: 4465-4474, 2003. suppressor genes have been associated with sensitivity and 2 Fu D, Calvo JA and Samson LD: Balancing repair and tolerance resistance, respectively, to cancer therapies (33). Many such of DNA damage caused by alkylating agents. Nat Rev Cancer genes code for proteins that are implicated in regulation of the 12: 104-120, 2012. cell cycle, such as the products of TP53, p21, transcription 3 Spanswick VJ, Craddock C, Sekhar M, Mahendra P, factor E2F (E2F), and retinoblastoma tumor suppressor (PRB), Shankaranarayana P, Hughes RG, Hochhauser D and Hartley JA: Repair of DNA interstrand crosslinks as a mechanism of whose dysregulation can have a major influence on the response clinical resistance to melphalan in multiple myeloma. Blood to cytotoxic agents because of their effects on tumor cell 100: 224-229, 2002. apoptosis and survival (34). Notably, loss of the TP53 gene has 4 Krause C, Kluttermann K and Mauz-Korholz C: Molecular been associated with resistance to multiple agents; however, at mechanisms and gene regulation of melphalan- and hyperthermia- least one large screen failed to discern this association, perhaps induced apoptosis in Ewing sarcoma cells. Anticancer Res 28: because the near universality of p53 inactivation in cancer lines 2585-2593, 2008. rendered this feature undifferentiating (33, 34). A detailed 5 Kresse M, Latta M, Kunstle G, Riehle HM, van Rooijen N, Hentze H, Tiegs G, Biburger M, Lucas R and Wendel A: Kupffer molecular definition of the cell lines used in our study is beyond cell-expressed membrane-bound TNF mediates melphalan the scope and intent of this discussion; however, we too have hepatotoxicity via activation of both TNF receptors. J Immunol not observed any correlation between mutations in common 175: 4076-4083, 2005. tumor suppressor genes such as TP53 and adenomatous 6 Fojo T and Menefee M: Mechanisms of multidrug resistance: polyposis coli (APC) and resistance to drug-induced apoptosis. The potential role of microtubule-stabilizing agents. Ann Oncol For example, the colorectal cancer cell lines Colo 320 HSR and 18(Suppl 5): v3-8, 2007. HCT-15 contain mutations in the TP53 and APC genes (Sanger 7 Perez EA: Microtubule inhibitors: Differentiating tubulin- inhibiting agents based on mechanisms of action, clinical Institute Core Cell Line Viewer, http://www.sanger.ac.uk), but activity, and resistance. Mol Cancer Ther 8: 2086-2095, 2009. the concentration of melphalan required to induce significant 8 Rodriguez-Antona C: Pharmacogenomics of paclitaxel. apoptosis is lower (4.83 μM and 5.05 μM respectively) than the Pharmacogenomics 11: 621-623, 2010. average (70.1 μM) observed for all colonic cell lines. A similar 9 Todd RC and Lippard SJ: Inhibition of transcription by platinum trend is seen for these two cell lines in the case of oxaliplatin- antitumor compounds. Metallomics 1: 280-291, 2009. induced apoptosis (see Tables II and IV). By contrast, the gastric 10 Makrilia N, Syrigou E, Kaklamanos I, Manolopoulos L and cancer cell line Hs 746T, for which the only reported mutation Saif MW: Hypersensitivity reactions associated with platinum antineoplastic agents: a systematic review. Met Based Drugs has been in the C-MET gene (35), was resistant to apoptosis 2010. induction by all three drugs tested. Thus, biomarker signatures 11 Wang Y, Probin V and Zhou D: Cancer therapy-induced that can be used to predict sensitivity and resistance to more residual bone marrow injury: Mechanisms of induction and general chemotherapeutics, such as those used in our study, may implication for therapy. Curr Cancer Ther Rev 2: 271-279, not be inherently obvious as mutations and may require more 2006. systematic approaches based on expression profiling. 12 van Iersel LB, Hoekman EJ, Gelderblom H, Vahrmeijer AL, van Persijn van Meerten EL, Tijl FG, Hartgrink HH, Kuppen PJ, Conclusion Nortier JW, Tollenaar RA and van de Velde CJ: Isolated hepatic perfusion with 200 mg melphalan for advanced noncolorectal liver metastases. Ann Surg Oncol 15: 1891-1898, 2008. Our results clearly demonstrate that melphalan, oxaliplatin, and 13 Alexander HR, Libutti SK, Bartlett DL, Puhlmann M, Fraker DL paclitaxel are good candidates for further study in the CS-PHP and Bachenheimer LC: A phase I-II study of isolated hepatic system. These agents, at concentrations that are feasible using perfusion using melphalan with or without tumor necrosis factor CS-PHP, induced significant apoptosis in liver, colonic, and for patients with ocular melanoma metastatic to liver. Clin gastric cancer cells but were not toxic to primary human Cancer Res 6: 3062-3070, 2000. 14 Alexander HR Jr., Libutti SK, Pingpank JF, Steinberg SM, hepatocytes, even at high doses and extended exposure times Bartlett DL, Helsabeck C and Beresneva T: Hyperthermic of 1 and 4 h. Thus, while cell line testing in this format is not isolated hepatic perfusion using melphalan for patients with always indicative of actual results in humans, this study ocular melanoma metastatic to liver. Clin Cancer Res 9: 6343- provides compelling evidence that the compounds tested could 6349, 2003.

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