ANTICANCER RESEARCH 37 : 6575-6581 (2017) doi:10.21873/anticanres.12114 Paclitaxel, Carboplatin and 1,25-D3 Inhibit Proliferation of Endometrial Cancer Cells In Vitro TEA KUITTINEN 1, PÄIVI ROVIO 1, SYNNÖVE STAFF 1,2 , TIINA LUUKKAALA 3,4 , ANNE KALLIONIEMI 5,6 , SEIJA GRÉNMAN 7,8 , MARITA LAURILA 9 and JOHANNA MÄENPÄÄ 1,10 1Department of Obstetrics and Gynaecology, Tampere University Hospital, Tampere, Finland; 2Laboratory of Cancer Biology, BioMediTech Institute, Faculty of Medicine and Life Sciences, University of Tampere, Tampere, Finland; 3Research and Innovation Centre, Tampere University Hospital, Tampere, Finland; 4Health Sciences, Faculty of Social Sciences, University of Tampere, Tampere, Finland; 5BioMediTech Institute and Faculty of Medicine and Life Sciences, University of Tampere, Tampere, Finland; 6Fimlab Ltd, Tampere University Hospital, Tampere, Finland; 7Department of Obstetrics and Gynaecology, Turku University Hospital, Turku, Finland; 8University of Turku, Turku, Finland; 9Department of Pathology, Fimlab Ltd, Tampere University Hospital, Tampere, Finland; 10 Faculty of Medicine and Life Sciences, University of Tampere, Tampere, Finland Abstract. Background/Aim: Endometrial cancer cells are combination of these compounds. The corresponding numbers known to be sensitive to carboplatin and paclitaxel. in UT-EC-3 were 70%, 33% and 65%, respectively. 1,25-D3 Further more , vitamin D (1,25-D3) has been reported to inhibit suppressed cell growth 88% with paclitaxel, 63% with endometrial cancer cell growth both as a single agent and carboplatin and 87% with their combination in the UT-EC-1 combined with carboplatin. However, there are no studies cell line. Conclusion: In both cell lines, single-agent paclitaxel comparing the effect of paclitaxel and carboplatin as single was as effective as the combination of the compounds and agents vs. in combination in endometrial cancer cell lines. more effective than single carboplatin. 1,25-D3 may further Neither has the effect of 1,25-D3 been studied with paclitaxel. contribute to the cytotoxic effect of these agents. The present study investigated the effect of paclitaxel, carboplatin and 1,25-D3 on the growth of endometrial cancer Endometrial cancer is the most common gynecological cells in vitro. Materials and Methods: Two endometrial malignancy in developed countries. In Finland incidence is adenocarcinoma cell lines (UT-EC-1 and UT-EC-3) were 13.9 per 100,000 persons a year (1). Most women are cultured with different doses of paclitaxel, carboplatin and diagnosed with early-stage disease and have a high cure rate 1,25-D3. The cellular VDR (vitamin D receptor) mRNA levels with surgery alone with a reported 5-year survival rate of were measured and the expression of estrogen (ER) and 90% (2). Still up to 25% of women will have advanced, high progesterone (PR) receptors by the cells was determined. grade or recurrent disease and require additional treatment. Results: In the UT-EC-1 cell line the growth inhibition was In these cases, the therapeutic response to cytotoxic and 72% with paclitaxel, 54% with carboplatin and 73% with the hormonal agents has been typically only partial and of short duration. There is no generally accepted standard chemotherapy in treatment of advanced and recurrent endometrial carcinoma but simultaneous administration of This Αrticle is freely accessible online. carboplatin and paclitaxel or carboplatin and pegylated liposomal doxorubicin are widely used (3-8). Correspondence to: Tea Kuittinen, Department of Obstetrics and Vitamin D is an antiproliferative and immunomodulatory Gynaecology, Tampere University Hospital, Teiskontie 35, 33520 secosteroid hormone with a well-established role in Tampere, Finland. Tel: +3585031169810, e-mail: tea.kuittinen@ xfimnet.fi maintenance of calcium synthesis. Active metabolite of vitamin D, 1alpha,25-dihydroxyvitamin D 3 (1,25-D3) binds Key Words: Vitamin D, 1,25-D3, endometrial cancer in vitro , to the vitamin D receptor (VDR) in the cell nucleus. The paclitaxel, carboplatin, growth inhibition, VDR. function and/or expression of the VDR is needed for the 6575 ANTICANCER RESEARCH 37 : 6575-6581 (2017) growth regulatory effects of 1,25-D3 in vitro and disabled used were 1-100 nM and fresh stock solutions were made for each VDR activity leads to 1,25-D3 insensitivity and accelerated experiment. Carboplatin was diluted in sterile water to get a solution tumorigenesis (9). Many different cell types, including of 100 μg/ml. The final dilutions used were 0.1-20 μg/ml. The dilutions were made immediately before use. 1,25-D3 was diluted in normal and malignant endometrial cells, contain VDR and DMEM to get final 0.1-100 nM solutions. The concentrations of 1alpha hydroxylases to synthesize active 1,25-D3 locally (10, drugs were based on previous reports as well as physiological 11). This active metabolite does not enter the circulation and relevance (23, 24, 27, 28). has no effect on calcium homeostasis, but is rather degraded by 24-hydroxylases. VDR, ER and PR expression analyses. The VDR mRNA levels in UT- 1,25-D3 is well known for its antiproliferative roles EC-1 and UT-EC-3 cells were measured by qRT–PCR using the LightCycler equipment (Roche, Mannheim, Germany). Total RNA including the induction of G 0-G 1 cell-cycle arrest with was extracted using the RNeasy Mini Kit (Qiagen, Valencia, CA, associated down-regulation of oncogenic cyclins D1 and D3 USA) and was reverse transcribed with SuperScript™ First-Strand and p27/p21 induction (12-15). 1,25-D3 is responsible for Synthesis System for RT–PCR (Invitrogen, Carlsbad, CA, USA) as VDR-mediated apoptosis and inhibits angiogenesis and described (29). qRT-PCR was performed using 20 μM gene specific differentiation in a variety of cancer types (13, 16-20). 1,25- primers (Sigma-Aldrich, sense 5’ ATCGGCATGATGAAGGAGTT D3 is a potential antitumor agent because of its ability to 3’, antisense 5’ TGCTCCTCAGACAGCTTGG 3’) and 10 μM UPL regulate cancer cell growth. 1,25-D3 has been found to probe (probe number # 12). The PCR program included the following inhibit endometrial cancer cell growth both as a single agent steps: 10 min denaturation at 95˚C followed by 45 cycles of 10 s denaturation at 95˚C, 30 s annealing at 60˚C and 1 s elongation at (21) and combined with carboplatin (22). 72˚C. The expression levels were normalized using glyceraldehyde- Endometrial cancer cells are sensitive to carboplatin, 3-phosphate dehydrogenase (GAPD) housekeeping gene. cisplatin and paclitaxel (23, 24). Still there are no studies The expression of estrogen (ER) and progesterone (PR) receptors comparing paclitaxel and carboplatin as single agents vs. in in both cell lines was determined with immunohistochemical combination in endometrial cancer in vitro or in vivo . In staining. ER and PR immunostainings were performed using addition, there are no studies examining the effect of vitamin Ventana Benchmark Ultra automated IHC/ISH Slide staining system D with paclitaxel or the combination of paclitaxel and (Ventana Medical Systems, AZ, USA). Deparaffinization and antigen retrieval were performed using Roche Ventana reagents (EZ carboplatin on endometrial cancer cells. The aim of the Prep 10X concentrate and Cell Conditioning Solution CC1). ER present study was to investigate the potential effects of staining was performed using a Roche Ventana RTU Confirm anti- paclitaxel, carboplatin and 1,25-D3 on the growth of Estrogen Receptor Rabbit monoclonal antibody (Clone SP1) with endometrial cancer cells both as single agents and in 32 min incubation time. PR staining was performed using a Roche different combinations with each other in vitro . Ventana RTU Confirm anti-Progesterone Receptor Rabbit monoclonal antibody (Clone 1E2) with 24 min incubation time. Materials and Methods Both immunostainings were detected and visualized using Roche Ventana UltraView DAB IHC Detection Kit. Cell lines. Two endometrial adenocarcinoma cell lines were used Cell growth assays. For each experiment, the cells were plated on (UT-EC-1 and UT-EC-3) in this study. The cell lines were originally 24-well plates at 30,000 cells/well. Cells were then treated with established from primary tumors before any treatment using an indicated doses of paclitaxel (1-100 nM), carboplatin (0.1-20 μg/ml) explant technique (25, 26). UT-EC-1 was established from a and vitamin D (0.1-100 nM) alone or in combination. After a 3- curettage specimen of a clinical FIGO Stage II Grade 2 endometrial 3 day incubation period, the cells were trypsinized and counted using papillary adenocarcinoma and UT-EC-3 from the hysterectomy the Z2 Coulter Counter (Beckman Coulter, Fullerton, CA). All specimen of a surgical FIGO Stage Ia Grade 3 endometrial experiments were done in six replicates and were repeated at least adenocarcinoma (23). The biological properties of these cell lines twice. have been described in detail earlier (23). Statistical analyses. Non-parametrical Mann-Whitney test was used to Cell cultures and treatments. Prior to the experiments the cells were assess differences between treated and control groups. Exact p- values cultured as described previously (23). In brief, the cells were plated under 0.05 were considered as statistically significant. All statistical weekly and maintained in logarithmic phase in 75 cm 2 culture flasks analyses were performed using IBM SPSS Statistics version 23 (IBM in Dulbecco’s modified Eagle minimal essential medium (DMEM), SPSS Statistics, IBM Corporation, Chicago, IL). supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, 1% glutamine and 1% non-essential amino acids at 37ºC in a humidified atmosphere containing 5% CO 2. Both Results cell lines were tested negative for mycoplasma contamination. Paclitaxel (Hospira ® 6 mg/ml, Maidenhead, UK) and carboplatin The cells were first cultured with different concentrations of (Accord ® 10 mg/ml, Middlesex, UK) were provided by the Pharmacy of the Tampere University Hospital. 1,25-D3 was paclitaxel (1, 3, 5, 10, 50, and 100 nM) and carboplatin (0.1 purchased from Sigma-Aldrich (St.