A Potent Chemotherapeutic Strategy With Eg5 Inhibitor Against Gemcitabine Resistant Bladder Cancer

BackgroundBladder cancer is the most common genitourinary tumor in China. Approximately 25% of bladder cancer patients are diagnosed with muscle-invasive bladder cancer (MIBC), although 75% of newly diagnosed tumors are non-muscle invasive (Ta, Tis, and T1); most of them recur and 15-20% progress to invade tunica muscularis. And the vast majority of cancer-specific deaths are due to MIBC, leading to local invasion and distant metastasis. The mortality of the disease urges urologists to explore novel methods to treat bladder cancer. Chemotherapy with gemcitabine and cisplatin is the most popular option for bladder cancer. But the occurrence of chemoresistance greatly limits the long-term curative effect.Gemcitabine is an analog of deoxycytidine with high activity against many types of solid tumors including pancreatic, cervical, ovarian, breast, bladder, and non-small cell lung cancers. However, the development of resistance to gemcitabine is now a major concern to urologists. Despite a reasonable response rate after initial chemotherapy in patients with metastatic bladder cancer,60-70% of responding patients relapse within the first year, with a median survival of 12-14 months. This limited efficacy may be due to de novo drug resistance and the development of cellular drug-resistant phenotype during treatment.However, the underlying mechanisms of inducing chemotherapy resistance by gemcitabine remain unknown. Recently, through the study of pancreatic cancer, Nakahira S et al reported an important factor in gemcitabine resistance was the overexpression of ribonucleotide reductase (RR). RR consists of the dimerized large and small subunits, M1 and M2, respectively. The Ml subunit possesses a binding site for enzyme regulation (regulatory subunit), and the M2 subunit is involved with RR activity (catalytic subunit). RRM1 is supposed to play a role in gemcitabine resistance of the variety of cancer as metabolic enzymes of the drug. RRM1 is not only a cellular target for gemcitabine, but also a tumor suppressor. Preclinical studies have demonstrated its involvement in the suppression of cancer cell proliferation, migration, and metastasis. In some cancers, a high level of RRM2 mRNA correlates with chemotherapeutic resistance, cellular invasiveness and unsatisfied prognosis, suggesting that RRM2 contributes to malignant progression and is a potential therapeutic target. However, there is limited information concerning RRM1 and RRM2 protein expression in bladder cancer, and to our knowledge no reports exist describing the role of RRM in the process of drug resistance in bladder cancer. Moreover, some recent studies have indicated that RRM plays an important role in the development and progression of human carcinomas, but the clinical significance of RRM expression in BCa remains unclear.On the other hand, it is of great significance to investigate novel bladder cancer chemotherapeutic strategy. Targeted drugs in the treatment of urinary tract tumors in recent years showed promising results. But so far there was no RRM related targeted drugs provided.Our early studies have found that Eg5 inhibitors as targeted drugs in vivo and in vitro treatment of prostate cancer and bladder cancer should have satisfying curative effects. Eg5, a key molecule involved in the formation of bipolar spindles, is one of the most attractive target enzymes in antimitotic drug discovery. Eg5 accounts for many of the movements of the spindle and chromosomes in dividing cells and localizes to the spindle in mitotic dividing cells. An interesting feature of Eg5 is that it localizes to microtubules in mitosis, but not to interphase microtubules, suggesting that an Eg5 inhibitor may be useful to specifically target proliferating tumor tissue. Several chemical types of small molecule Eg5 inhibitors have been reported. S-(4-methoxytrityl)-L-cysteine (S(MeO)TLC), a derivative of S-trityl-L-cysteine (STLC), can specifically inhibit Eg5, and induce monopolar mitotic spindle formation. Failure of Eg5 function leads to cell cycle arrest in mitosis with monoastral microtubule arrays. The important role of Eg5 in mitotic progression makes it an attractive candidate for developing targeted therapy in cancer. However, there is no study of Eg5 inhibitor treatment of Gemcitabine resistant bladder cancer.ObjectiveWe investigate the relationship between RRM1 and RRM2 expression and prognosis of patients with gemcitabine-resistant bladder cancer, and then examine the anticancer activity of S(MeO)TLC for gemcitabine-resistant bladder cancer in vivo and in vitro.MethodsGemcitabine-resistant cells were developed by chronic, repeated exposure to gemcitabine though gradient culture. IC50 of gemcitabine-resistant cells to gemcitabine was assessed using MTT assay, microarray analysis were used to analyze the difference between RT112-Gr and RT112. RRM1 and RRM2 expressions in bladder cancer cell lines were determined by RT-PCR analysis. To assess the effects of RRM1 and RRM2 on RT112-Gr bladder cancer cell lines,RRMl/2 gene were Knockdown, respectively.RRM1 and RRM2 expression was examined by immunohistochemistry in bladder cancer specimens, and the correlation between clinicopathological characteristics and RRM1 and RRM2 expression was evaluated. The prognostic significance of RRM1 and RRM2 immunoreactivity was analyzed via survival analysis in 108 bladder cancer cases that were treated with adjuvant gemcitabine chemotherapy after surgery.To find co-expression gene with RRM1 and RRM2 in bladder cancer, We use cBioPortal software (http://www. cbioportal. org/public-portal/) to analyze The Cancer Genome Altas (TCGA) database.The anti-proliferative activity of S(MeO)TLC was analyzed in RT112-Gr bladder cancer cell lines using MTT and assay. The anticancer efficacy of S(MeO)TLC was investigated in vitro using an apoptosis assay with Hoechst nuclear staining and flow cytometry. Western blot were used to elucidate the inhibitory mechanism. Furthermore, the inhibitory effect was evaluated in vivo using subcutaneous xenografts models.ResultsRT112-Gr cells were 350-fold less sensitive to gemcitabine than the parental cell lines, which IC50 was 4.2 umol/1 in former. Of the 55752 genes represented on the Human OneArray(?) GeneChip,442 showed significantly up-regulated and 235 down-regulated, and the number of genes clustered was 227. RT-PCR further demonstrated that RT112-Gr cells had significant increases in the levels of RRM1 and RRM2 mRNA compared with the parental cells (P<0.001), respectively. Our study indicated that after Knockdown of RRM1 and RRM2, RT112-Gr bladder cancer cell lines sensitive to gemcitabine again.The expression of RRM1 was significantly associated with tumor size (P=0.023) tumor stage (P=0.048) and grade (P=0.01). The expression of RRM2 was significantly associated with tumor size (P=0.004), tumor stage (P=0.018) and grade (P=0.008).108 patients with bladder cancer were regularly followed up with the mean of 37.8 (from 5 to 79) months. The Kaplan-Meier survival curves demonstrated that Overall survival (OS) of the RRM1 and RRM2 high expression group was significantly shorter than the RRM1 and RRM2 low expression group (P= 0.022, P=0.016). The multivariable analyses demonstrated that the stage, grade, RRM1 and RRM2 reactivity were identified as independent prognostic predictors with adverse significance in patients with BCa (P=0.005, P ＜0.001, P= 0.045, P=0.033).Through analyze The Cancer Genome Altas (TCGA) database, we find RRM1 and RRM2 gene is co-expression with Eg5 (KIF-11) in bladder cancer. The IC50s of RT112 and RT112-Gr against S(MeO)TLC were 210μM and 290μM, respectively. S(MeO)TLC displayed the most prominent suppression character in RT112-Gr bladder cancer cell lines, and induced cell death after mitotic arrest via the Caspase-dependent apoptotic pathway. In vivo, it was found that S(MeO)TLC(20mg/kg) +gemcitabine(50mg/kg) and S(MeO)TLC (20mg/kg) groups prominently suppressed tumor growth in comparison with gemcitabine(50mg/kg) and control.ConclusionOur data have shown that RRM1 and RRM2 may associated with gemcitabine resistance. RRM1 and RRM2 overexpression is an important novel prognostic factor in patients with bladder cancer who received gemcitabine-based therapy, and the enhanced expression of RRM1 and RRM2 associates with poor prognosis. RRM1 and RRM2 reactivity were identified as independent prognostic predictors with adverse significance in patients with BCa. RRM1 and RRM2 gene is co-expression with Eg5 (KIF-11) in bladder cancer. S(MeO)TLC, as a novel Eg5 inhibitor, exhibited significant anticancer efficacy in gemcitabine resistant bladder cancer cell lines both in vitro and in vivo, and may provide a new therapeutic option to overcome chemoresistance in bladder cancer.