DR TAKATORI ERIKO Anticancer agents

Japan-based researcher Dr Eriko Takatori discusses how her research is advancing scientific understanding of the drug-induced DNA damage of tumour cells in clear cell carcinoma of the ovary, leading to the development of improved therapies, and potentially, individualised treatment

currently ongoing. We examined these four paradigms in the field of ovarian ? drugs in our research. We received our research equipment and our Can you explain the antitumour activity grant from the University. Importantly, co- of the anticancer platinum agents CDDP, workers helped us to collect clinical samples CBDCA, PTX and SN-38? and we were able to draw on the expertise of other researchers in the institution, who As platinum agents, CDDP and CBCDA gave some excellent advice about various inhibit DNA synthesis by causing the elements of our work. Additionally, the cell crosslinking of DNA. Moreover, CDDP and lines we used were obtained from another CBCDA also produce free radicals that institution in Japan where ovarian cancer damage DNA. PTX induces the formation research is being conducted. of unusual bundles, stabilises and inhibits microtubule Have your findings to date provided you depolymerisation by promoting tubulin with any new research avenues? polymerisation, thereby inhibiting DNA synthesis. SN-38 is the active metabolite of Our data showed that the effect of CPT-11 – and it works as a I antitumour drugs on DNA damage or the Could you outline your research into the inhibitor, therefore inhibiting DNA synthesis. varied between the different drugs, differences in incidences and types of DNA even when the same species of cell line was damage induced by antitumour agents for How are you exploiting cell cultures, used. We are now investigating the reasons clear cell carcinoma (CCC) of the ovary? immunohistochemistry and flow that underpin these differences. In particular, cytometry in your investigation? we are planning to examine the expression of Many antitumour drugs work by inducing cell cycle-related proteins – such as p53, p21, primary or secondary damage to the We used two CCC cell lines. After exposure p16 and p27 cyclin E – and DNA repair-related DNA of the cancer cell. However, when it to the drugs with various concentrations proteins – such as ATM ATR, Chk1 and Chk2 – comes to the treatment of ovarian CCC, and at different times, the cells were in order to identify the different mechanisms the mechanisms of DNA damage induced collected and fixed. Following this,γ H2AX of cell response. Moreover, we are also by antitumour drugs are not clear. In view and DNA were stained and the DNA and investigating the optimal administration of this, our research focuses on whether γH2AX contents were measured using flow method – that is, single or combined antitumour drugs used in the treatment of cytometry. The γH2AX in each cell cycle was administration – of the antitumour drugs. CCC have distinct action mechanisms and determined, thereby allowing us to examine whether these potential differences might be the relationships between cell kinetics Can you summarise the project’s most associated with drug resistance. and DNA damage induced by antitumor significant achievements to date? agents. This method, called the γH2AX-FC Furthermore, how do you hope to see What anticancer agents are used for assay, can also be detected by an your research impacting on future treating ovarian cancer and how are they based on the difference in the amount of treatment strategies? contextualised within your research? γH2AX. Altogether, this excellent assay allows the evaluation of the cell cycle, DNA In comparison to the comet assay, the high (CBDCA) and (PTX) damage and apoptosis, all of which are very sensitivity of the γH2AX-assay enables us are the key drugs used for treating ovarian important for identifying the mechanisms of to detect DNA damage induced by even cancer. PTX with CBDCA therapy is a DNA damage induced by antitumour drugs. very low doses of a drug and, in addition, first-line treatment but CCC shows poor to evaluate the relationship between chemo-sensitivity. Due to this, 7-ethyl-10- To what extent has collaboration within the DNA damage and cell cycle control. Taken [4-(1-piperidino)-1-piperidino] carbonyloxy Department of Obstetrics and Gynaecology together, this simple and rapid assay could (CPT-11) and (CDDP) at Iwate Medical University School of pave the way for the implementation of combination therapy has been proposed Medicine, Japan, facilitated your research individualised medical treatment for patients for patients with CCC, with clinical trials and fostered innovation in establishing new with refractory CCC.

102 INTERNATIONAL INNOVATION DR ERIKO TAKATORI

Combating clear cell carcinoma

The Department of Obstetrics and Gynaecology in Iwate Medical University School of Medicine, Japan, hosts a group of researchers who focus their research efforts on investigating antitumour drug mechanisms for one of the deadliest forms of ovarian cancer

OVARIAN CANCER IS a lethal gynaecological aim of our study was to examine the optimal malignancy that is most common in women method of administration and effective drug who have had the menopause. At present, combinations for the treatment of ovarian the current standard treatment for all of CCC,” outlines Dr Eriko Takatori, one of its different subtypes involves the surgical the leading researchers in the Department. removal of the tumour, followed by Paclitaxel Together, Takatori and her team have (PTX) and carboplatin (CBDCA) combination analysed the activities of four anticancer . However, in spite of progress agents, including CBDCA and PTX, as well as in surgical techniques and chemotherapy over 7-ethyl-10-[4-(1-piperidino)-1-piperidino] the past few decades, the five-year survival rate carbonyloxy camptothecin (CPT-11) and of patients who present with advanced stages cisplatin (CDDP). of the disease remains low, at approximately 40 per cent – a trend that is thought to be the MAPPING THE METHODS consequence of a lack of effective therapies. This poor survival rate is even more pronounced In their project, Takatori and her team in clear cell carcinoma (CCC) of the ovary, investigated differences in the incidences and with studies highlighting that this subtype of types of DNA damage caused by the four epithelial ovarian cancer is the least responsive antitumour agents for CCC of the ovary. They to chemotherapy. Indeed, while response used two ovarian CCC cultured cell lines to rates to combination chemotherapy in both explore these differences – namely, OVISE serous adenocarcinoma and endometrioid and RMG-I – which they obtained from the adenocarcinoma are roughly 75 per cent, Health Science Research Resources Bank in response rates for ovarian CCC range between Osaka, Japan. Following culture, the cell lines 18 and 50 per cent. were treated with each of the antitumour agents and the cells were then collected, The World Health Organization (WHO) fixed and reacted with γH2AX – that is, the has defined CCC of the ovary as lesions phosphorylated histone H2AX. The next step characterised by clear cells growing in solid, involved detecting the presence of γH2AX in tubular or grandular patterns and hobnail cells each cell cycle phase through flow cytometry lining tubules and cysts. Research demonstrates – a laser-based, biophysical technology used that the incidence of CCC of the ovary has for cell counting and cell sorting and for the been increasing over time, accounting for detection of biomarkers – using fluorescein some 25 per cent of ovarian malignancies isothiocyanate (FITC) and propidium iodide. in Japan and between 5 and 6 per cent in Europe. Its prevalence in Japan is thought to Importantly, recent research has shown that be due to the fact that Japanese women have phosphorylation of the histone H2AX – a the highest levels of endometriosis in the variant of the core histone H2A – is an extremely world – a condition that is closely associated sensitive and accurate marker of specific DNA with ovarian CCC. Surgical treatment is not a damage: “When DNA damage occurs, serine first-line therapy for endometriosis in Japan, 139 of histone H2AX – in chromatins on both potentially causing CCC tumours in the ovary sides of double-strands breaks (DSBs) – is to be overlooked. phosphorylated,” Takatori discloses. “Dot γH2AX is detectable using the γH2AX specific Scientists in the Department of Obstetrics antibody and therefore DNA damage can be and Gynaecology at Iwate Medical University immunocytochemically detected and visible. School of Medicine are taking the rising In addition, using flow cytometric bivariate prevalence of ovarian CCC seriously. They analysis of γH2AX and DNA contents, γH2AX have been conducting detailed investigations in each cell cycle can be determined, thereby into the different mechanisms of DNA allowing us to examine the relationships damage induced by antitumour drugs used between cell kinetics and DNA damage, which in the treatment of CCC of the ovary: “The have been induced by antitumour agents.”

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APPLICATION TO INDIVIDUAL A PIONEERING PROJECT TREATMENT AND ELUCIDATION OF Takatori’s project was the very fi rst study in Japan to examine the antitumour mechanisms ANTICANCER DRUG MECHANISM OF by looking at the relationship between the cell cycle and DNA damage. Using the ACTION WITH H2AX, AND THERAPY phosphorylated histone H2AX as an assay, DNA damage was identifi ed to a high degree of EVALUATION sensitivity even at very low concentrations. OBJECTIVE To improve the prognosis of clear cell carcinoma (CCC). This is achieved by FAR-REACHING FINDINGS conducting fl ow cytometric bivariate analyses of γH2AX and DNA contents in The results from Takatori’s study suggest two different cell lines of CCC (OVISE and that a combination of agents arresting the RMG-I) treated with cisplatin (CDDP), cell cycle – in turn causing the accumulation carboplatin (CBDCA), paclictaxel (PTX) of cells in the S-phase and inducing DNA or SN-38, and examining the effects of damage in S-phase cells – could be an these drugs with regard the induction of effective treatment for CCC of the ovary DNA damage, apoptosis and cell-cycle with a slow growth rate and a low ratio of progression vis-à-vis the cell-cycle phase. S-phase cells. Specifi cally, the fi ndings imply that the most effective treatment for CCC KEY COLLABORATORS of the ovary is likely to be combination Dr Toru Sugiyama, Iwate Medical chemotherapy of CDDP plus CPT-11 or GEM University School of Medicine plus CBDCA.

Dr Akira Kurose, Hirosaki University School Indeed, using their γH2AX assay the of Medicine researchers examined the relationships This is a representative microscopic image of OVISE CONTACT between antitumour effects and cell cycle cell line after exposure to 10 µg/ml cisplatin (CDDP) perturbations rapidly and to a very high for 24 hours. γH2AX foci and nuclear are stained Dr Eriko Takatori degree of precision. Promisingly, this means green and red, respectively. Principal Investigator that their innovative method has the ability to pinpoint the agents that are most effective Moreover, another drug under investigation Department of Obstetrics and Gynecology, in combating ovarian CCC in a relatively is everolimus, an allosteric inhibitor of Iwate Medical University School of Medicine short amount of time. mammalian target of rapamycin (mTOR) 19-1 Uchimaru that is currently used to treat advanced Morioka kidney cancer, breast cancer and pancreatic Iwate 020-8505 FUTURE DIRECTIONS cancer. Encouragingly, preclinical Japan Looking ahead, Takatori and her colleagues experiments have demonstrated that it T +81 19 651 5111 are planning to continue their research into can inhibit the proliferation of ovarian E [email protected] the impact of antitumour drugs on DNA cancer cells and increase their sensitivity damage and the cell cycle in CCC of the to cisplatin. However, it is unknown at http://kaken.nii.ac.jp/d/p/22591864. ovary. Excitingly, molecular-targeted drugs present how the mechanisms of PARP-1 or en.html are gaining leverage as a promising new mTOR inhibitors work when combined with strategy for the treatment of ovarian cancer: conventional in CCC of DR ERIKO TAKATORI is Assistant Professor “ – that is, a selective inhibitor the ovary, testifying to the need for further in the Department of Obstetrics and of poly-ADP-ribose polymerase-1 (PARP- studies in this area. Gynecology at Iwate Medical University 1) – has demonstrated antitumour abilities School of Medicine and a member of the in both breast cancer and ovarian cancer, Ultimately, the hope is that the research Japan Society of Obstetrics and Gynecology. with either breast cancer susceptibility gene conducted by Takatori and her team will DR TORU SUGIYAMA is Professor in the (BRCA1/2) ,” Takatori points out. lay the groundwork for new therapeutic Department of Obstetrics and Gynecology “Also, PARP-1 plays a major role in response strategies for CCC of the ovary, feeding at Iwate Medical University School of to extensive DNA damage in single-strand into the development of individualised Medicine. Sugiyama is also Director at Japan break (SSB) repair, so PARP-1 inhibitors treatments that improve the prognosis of Society of Gynecologic Oncology and Japan induce apoptosis in cancer cells with this deadly disease and, further down the Society of Clinical Oncology, and a delegate defi cient BRCA pathways.” line, save many lives. at the Japan Society of Obstetrics and Gynecology. These cytograms show DR AKIRA KUROSE is Professor in the bivariate distributions (DNA Department of Diagnostic Pathology at content vs γH2AX) of clear Hirosaki University School of Medicine. cell carcinoma (CCC) cell line, OVISE, treated with TAKATORI, SUGIYAMA AND KUROSE cisplatin (CDDP). OVISE was have authored over 300 peer-reviewed treated with 100 ng/ml, the publications. Their research is focused on minimum concentration inducing DNA damage that the investigation of ovarian CCC, particular was previously reported that shown to be chemo-resistant. They are on our study, for various dedicated to the improvement of prognosis reaction times. S-phase in patients with ovarian CCC. cells of OVISE showing DNA damage progressed to apoptosis after 48 hours. In addition, S-phase arrest was observed.

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