| BackgroundBladder cancer is the commonest malignancy of the urinary tract and the ninth most common cancer in the world, which is considered a chemo-sensitive disease[1]. CDDP-based combination therapies, such as the GC (CDDP and gemcitabine) and the MVAC (methotrexate, vinblastine, doxorubicin and CDDP) regimen have evolved as standard therapy for advanced or metastatic bladder cancer[2,3]. But the prognosis in patients with metastatic disease is still poor [3]. Despite a consistent rate of initial responses, CDDP treatment often results in the development of chemoresistance, leading to therapeutic failure. Therefore, chemosensitization by reversing the chemoresistance is a promising strategy with important clinical implications for bladder cancer therapy.CDDP exerts anticancer effects via multiple mechanisms, yet its best-characterized mode of action involves the generation of DNA lesions, followed by the activation of the DNA damage response (DDR)[4]. If limited in amount, the DNA lesions can be recognized and safely removed by several repair systems that normally operate in the context of a temporary cell cycle arrest at S and G2 phase. Conversely, when the DNA damage is beyond repair, either such a cell cycle arrest becomes permanent (known as cellular senescence) or cells become committed to die, most often via mitochondrial apoptosis[5-8]. These processes, involving cell cycle checkpoint, DNA repair and apoptosis, are referred as DDR. The activation of cell cycle checkpoint is essential for cell survival, for it arrests the cell cycle progress and allows time for DNA damage repair[9]. The ataxia telangiectasia and Rad3-related (ATR) protein kinase is a key enzyme in the DDR that activates checkpoint kinase 1 (Chkl), resulting in cell cycle arrest[11,46]. Following DNA damage, ATR binds to DNA damage site and phosphorylates and activates Chkl. The activated Chkl phosphorylates and inhibits CDC25 phosphatase, and thus resulting in cell cycle arrest to facilitate DNA repair and prevent transferring altered genetic information to daughter cells[5,10,46]. Besides, ATR can phosphorylate a class of substrates involved in DNA repair[11]. Therefore, targeting ATR/Chkl pathways may be an important strategy for enhancing the efficacy of DNA-damaging agents [10].Cdc6 is an essential licensing factor for DNA replication. The best-characterized function of Cdc6 is the assembly of pre-replicative complexes (pre-RC) at origins to initiates DNA replication in G1 phase[31]. Once the origins fire, Cdc6 is exported to the cytoplasm to prohibit rereplication. But a significant proportion of Cdc6 remains in the nucleus, suggesting a potential additional function(s) other than pre-RC assembly[32]. Recently, it is reported that human Cdc6 involves in the activation of ATR signal. Cdc6 physically interacts with ATR in a Cdk-phosphorylation-stimulated manner and that Cdc6 is required for the ATR-dependent replication-checkpoint response activated by modest replication stress[33]. Although Cdc6 is reported to be linked to cancer development[35-40], the exactly effects of Cdc6 on malignant progression in bladder cancer is still unknown. Cdc6 can physically interact with ATR, which functions as a master regulator of DNA damage response. Dysregulation of DNA damage repair contributes to chemotherapeutic resistance. We wonder if ATR-Chkl pathway is over-activated in cisplatin resistant bladder cancer cells and explore the function of Cdc6 in cisplatin resistant bladder cancer cells.Norcantharidin (NCTD), a demethylated analog of cantharidin, has been shown to exert a strong antitumor effect on many types of cancer. Cantharidin is the main ingredient for the anticarcinogenic effect of Mylabris, which has been used for antitumor treatment in traditional chinese medicine for centuries. NCTD causes fewer side effects on gastrointestinal and urinary tracts than Cantharidin. It is reported NCTD can cause cleavage of Cdc6 in Hela cells[Anticancer Drugs.2006 Mar;17(3):307-14]. So we investigated whether NCTD can lead to downregulation of Cdc6 in bladder cancer cells especially in cisplatin resistant cancer cells and enhance cancer-killing efficacy of cisplatin by inactivating ATR-Chkl checkpoint.Objectives, methods and resultsPart One Investigation on Expression pattern of Cdc6 in bladder cancer tissues and relationship between Cdc6 positivity and the clinical parametersIn the first part of study, in order to investigate Cdc6 expression pattern in bladder cancer and normal bladder tissue we compared Cdc6 protein expression in tumor tissues and normal bladder tissue of 12 patients by Western blot and analyzed the expression of Cdc6 in tissue chips from 105 bladder cancer patient using immunohistochemistry to further investigate the relationship between Cdc6 expression and clinicopathological characteristics in bladder cancer. Besides, we also analyzed Cdc6 expression in bladder cancer cell lines of T24, UMUC3 and 5637. The results show higher cdc6 expression in cancer tissues were found compared to its paired adjacent bladder tissues. Cdc6 positive expression were found in 81 out of 115 bladder cancer patient (70.4%), significant higher than in normal samples (6%,3 in 50, P<0.05). Positive-cdc6 expression correlated with tumor grade (low vs high grade,P=0.012). More positive-Cdc6 samples were detected in advanced tumor (T2-T4,75.6%) than in non-muscle-invasive tumor samples (Ta-T1,57.6%), but with no significant statistical differences (P=0.055). No statistical differences of Cdc6 expression were found according to age and gender. We next detected Cdc6 protein expression in bladder cancer cell lines UMUC3,5637, and T24 and found the strongest Cdc6 expression in UMUC3 cells.Part Two Investigation on effect of Cdc6 on DNA replication, migration and invasionCdc6 plays a major role in the formation and maintenance of pre-RC and acts as the rate limit to the initiation of DNA replication. Other researchers reported Cdc6 is over-expressed in many types of cancers. We found high expression of Cdc6 in bladder cancer tissues correlate with high tumor grade in bladder cancer. So we speculate Cdc6 may plays an crucial role in DNA replication, migration and invasion. In this study, we constructed small interfering RNA targeting Cdc6 and confirmed in capacity of silencing Cdc6. Brdu incorporation assays revealed that Cdc6 downregulation by siRNA can decrease Brdu positive cell proportion in UMUC3 and T24 cells, indicating Cdc6 depletion suppress DNA synthesis. Wound-healing assays demonstrated that Cdc6 RNAi significantly decreased migration capacity in UMUC3 cells. Similarly, Cdc6 depletion reduces cell number of migrating out of the upper chambers in T24 and UMUC3 cell lines. Furthermore, Cdc6 RNAi significantly reduced cell number invading through the ECM when compared to negative control. These results indicate that Cdc6 depletion can reduce the malignant traits, including DNA replication, migration and invasion of bladder cancer cells.Part Three Establishment of cisplatin- resistant cell line and investigation on the expression features of Cdc6 and ATR in cisplatin-resistant bladder cancer cells.Cisplatin resistance is the main obstacle to treat advanced bladder cancer. To investigate the mechanism of cisplatin resistance in bladder cancer cells, we select cisplatin resistant cancer cells from UMUC3 cells by long-term and interval exposure to low dose of cisplatin. After 90 days interval treatment by cisplatin, we obtained relatively cisplatin resistant cancer cells named as UMUC3R, and its low response to cisplatin was confirmed by MTS cell proliferation assays comparing cell viability of two types of cells treated by cisplatin. The results show UMUC3R cells exert lower sensitivity to cisplatin than UMUC3 cells with the extension of exposure time and increase of drug concentration. Cisplatin is the key ingredient in first line chemotherapeutic schemes and its main mechanism of killing cancer cells is leading to apoptosis by DNA damage.Therefore, it is supposed that efficient DNA repair capacity contribute to cisplatin resistance of UMUC3R cells. Once activated by DNA damage, ATR binds to loci of DNA lesion and phosphorylates a host of substrates, initiating a cascade that results in cell cycle arrest and DNA repair[11,12]. Based on crucial role of ATR in DNA damage response and interaction between Cdc6 and ATR, the mechanism of cisplatin resistance in bladder cancer may be related to overactive ATR pathway that promoted by Cdc6 that facilitates DNA repair. Therefore, we hypothesize that in CDDP-resistant bladder cancer cell, more ATR bind to the damaged DNA. Chromatin binding assays were used to evaluate chromatin-binding fraction of ATR and Cdc6 in UMUC3-R cells and its parent UMUC3 cells following CDDP treatment. The results show that low expression of ATR was detected without CDDP treatment. The protein levels were increased following CDDP treatment in a dose dependent manner both in UMUC3-R and UMUC3 cells. Notably, chromatin binding ATR in UMUC3-R was elevated greater than in non-resistant UMUC3 cells, indicating UMUC3-R cells have more powerful DNA repair capacity. On the contrary, Cdc6 in both chromatin and supernatant were decreased after CDDP treatment. As Cdc6 mainly function as DNA replication initiator, we deduce that the decreased Cdc6 protein level is attributed to the inhibitory effect of DNA replication induced by CDDP. However, it is noteworthy that the chromatin-binding Cdc6 protein levels in UMUC3R cells were remarkably higher than in UMUC3, especially after CDDP treatment. This suggests that Chromatin-binding Cdc6 in cisplatin resistant cells localize in nucleus more stably than in cisplatin-sensitive UMUC3 bladder cancer cells, hence promote ATR binding to DNA lesion loci and consequently enhance DNA repair capacity.Part four Impact of Cdc6 down regulation on apoptosis induced by cisplatinTo determine whether inhibition of Cdc6 can enhance the sensitivity of CDDP-resistant bladder cancer cells, Annexin V-FITC/PI stain apoptosis assays were done..CDDP (4μg/ml) induced 20% apoptosis in UMUC-3 cells versus about 9% in UMUC3-R cells, showing lower response to CDDP in UMUC3-R cells. The combination of CDDP with Cdc6 RNAi leaded to over 21% apoptosis in UMUC3-R and 40% in UMUC3 cells, which is significantly higher than apoptosis caused by CDDP alone, suggesting that Cdc6 RNAi synergy with CDDP promotes apoptosis both in CDDP-resistant and non-resistant cells. These results show Cdc6 may be a potent target to enhance antitumor efficacy by reversing resistance to cisplatin.Part Five Exploration on the mechanism of enhancing antitumor efficacy of cisplatin by Cdc6 RNAiActivated ATR-dependent-checkpoint pathway can arrest cell cycle progress to repair DNA. To understand whether Cdc6 depletion could break cell cycle arrest by inhibiting ATR checkpoint pathway, we examined the cell cycle distribution of UMUC3-R cells treated by CDDP, Cdc6 RNAi or combination of both. The results show CDDP resulted in S phase arrest (70% in S,14.6% in G2/M), while Cdc6 depletion caused a substantial proportion of cells abrogated S phase arrest and progressed into G2/M phase (56.7% in S,22.3% G2/M). Cell cycle analysis indicated that Cdc6 depletion led to abnormal progression of cancer cells into G2/M phase, under CDDP exposure. Here, to further characterize aberrant mitosis with CDDP-induced DNA damage, we assess pH3 (a mitotic marker) and yH2AX (a DNA damage marker) by immunofluorescence. The results showed few pH3-but plenty of yH2AX-stained cells in CDDP treatment group, while combination of Cdc6 RNAi with CDDP gave rise to more yH2AX-positive cells, indicating that Cdc6 depletion enhance DNA damage induced by CDDP. More importantly, more pH3 positive cells were observed in the combination group than in CDDP alone, and led to y-H2AX/pH3 double stained cells, suggesting aberrant mitosis was induced. Our studies show that Cdc6 depletion abrogated S phase block induced by CDDP and resulted in aberrant mitosis with DNA damage. So we speculate that inhibition of Cdc6 may impair activation of ATR-Chkl-CDC25C pathway and abolish cell cycle arrest, leaving DNA damage unrepaired. In accordance to previous reports, we found that CDDP can activate ATR-ChkI-Cdc25C pathway. Chromatin-binding ATR was increased after CDDP exposure, indicating ATR was loaded to the damage loci. Accordingly, the p-Chkl and p-CDC25C were also increased. As expectation, after Cdc6 RNAi transfection, Chromatin-binding ATR, p-Chkl and p-CDC25C were decreased, indicating that the CDDP-activated ATR-Chkl-CDC25C pathway was inhibited by Cdc6 depletion. These results indicate Cdc6 contributes to the activation of ATR-Chkl-Cdc25C pathway under DNA damage stress.Part Six Cdc6 downregulation by NCTD reverses cisplatin resistance in cisplatin-resistant bladder cancer subcutaneous tumor model in nude miceThe results of Western blot and immunohistochemistry demonstrated that Cdc6 are overexpressed in bladder cancer tissues and correlates with tumor grade of bladder cancer. Cdc6 depletion by siRNA can not only suppress proliferation, migration and invasion in bladder cancer cell lines, but also reverse cisplatin resistance by inhibiting activation of ATR-Chkl-Cdc25C pathway and abrogate cell cycle arrest induced by ATR dependent checkpoint pathway leading to aberrant mitosis with DNA damage. To determine whether Cdc6 depletion can overcome cisplatin resistance in vivo, nude mice subcutaneous transplantation tumor model was constructed. Our previous study show can lead to downregulation of Cdc6 in Hela and DU145 cells. Here, we confirmed norcantharidin can reduce Cdc6 expression in UMUC3R and UMUC3 bladder cancer cells. After subcutaneous transplantation tumor of cisplatin resistance UMUC3R were implanted, nude mice were randomly divided into norcantharidin combined cisplatin group, cisplatin group, norcantharidin group and control group. The results show tumor growth slow down in NCTD or CDDP single treatment group but tumor still grows, indicate either CDDP or NCTD cannot killing UMUC3R cisplatin resistance cancer cells alone effectively. Of note, the volume of subcutaneous tumor mass were markedly decreased in combination treatment group compared to single treatment group and control group. This results suggest that NCTD can enhance antiproliferative effects of cisplatin on cisplatin resistant UMUC3R cell in vivo thus indicating the potential for a combined therapy. To clarify the molecular mechanisms underlying NCTD-induced reversal of drug resistance, we analyzed Cdc6, ATR, gamma-H2AX expression in indicated groups by Immunohistochemistry. Cdc6 expression in NCTD is significantly lower than in control group, indicating NCTD can reduce Cdc6 expression in vivo. In CDDP group, Cdc6 stain is weaker compared to control group, but considerable amount of Cdc6 still was detected in the nucleus of UMUC3R cells, showing stable localization of Cdc6 in nucleus under CDDP treatment. In NCTD combined cisplatin group, Cdc6 stain is weakest and ATR expression is lower than in CDDP treatment group, indicating downregulation of Cdc6 cannot facilitate activation of ATR pathway to arrest cell cycle which is required for DNA repair. Stronger gamma-H2AX stain in combination group compared with that in CDDP group states that NCTD can enhance the efficacy of CDDP in damaging DNA. To sum up, combined treatment of NCTD and cisplatin may provide a new therapeutic option to enhance chemosensitivity in cisplatin resistant bladder cancer and the underlying mechanism may be attributed to that low expression of Cdc6 caused by NCTD treatment suppressed activation of ATR dependent DNA repair pathway and consequently lead to more severe DNA damage to cisplatin resistant bladder cancer cells.ConculsionIn this study, we found elevated Cdc6 expression in bladder cancer tissues compared to normal bladder tissues. There are significant correlation between Cdc6 up-regulation and higher tumor grade. These results are consistently with previous researches in other type of cancers, such as oral squamous cell carcinoma[39], lung carcinomas[36,47], cervical carcinoma[48], gallbladder carcinoma[37], prostate cancer[49]. Considering that Cdc6 is normally absent in quiescent and differentiated cells, it could be specific markers for cancer cells. In this research, CDDP-resistant bladder cancer cells shows increased chromatin-binding ATR and Cdc6 compared to parent cells. Furthermore, Cdc6 depletion not only can inhibit DNA replication and invasion, but also reverse CDDP resistance of UMUC3R and cause abnormal mitosis, probably by inactivation of ATR-Chk1-Cdc25 pathway. These results indicate that Cdc6 contributes to malignant progression of bladder cancer, and could be used as a potential anticancer target.Cdc6 inhibition can abolish S/G2 checkpoint and induce abnormal mitosis with DNA damage indicating silencing Cdc6 might be cytotoxic to tumors as well as to normal cells. However, the intact G1 and S/G2 cell cycle checkpoints should give survival advantage for normal cells. The ataxia telangiectasia-mutated (ATM) and ATR are the two major signal transducers in DNA damage response. Cancer cells lack G1 checkpoint because of deficiency in ATM/p53 signaling[14-19]. It has been hypothesized that cancer cells rely solely on the ATR/Chkl pathway and S/G2 checkpoints for repairing DNA damage[20,22-27]. Therefore, ATR inhibition in tumor cells can lead to accumulation of cytotoxic damage and lethal mitotic catastrophe, whilst normal cells with a functional G1 checkpoint coordinating DNA repair and cell cycle arrest are unaffected[28-30][54-57]. Therefore, targeting ATR/chkl pathways is a useful strategy for enhancing the cancer-selective killing efficacy of DNA-damaging agents. On the ground of the multiple roles in cell DNA replication and ATR-Chkl checkpoint, Cdc6 may be an efficient target with unique advantage.In conclusion, in our present study, we first report that Cdc6 is upregulated in bladder cancer tissues and is correlated with tumor grade. More importantly, Cdc6 promotes CDDP resistance in bladder cancer cells by collaborating with ATR signal pathway. Accordingly, inhibition of Cdc6 could enhance cytotoxicity of CDDP in both parent and CDDP-resistant bladder cancer cells. Considering the deficiency of ATM-dependent pathway in cancer cells and the important role of Cdc6 and ATR in chemo-resistant cancer cells, Cdc6 may be a potential specific therapeutic target for cancer, especially for CDDP-resistant cancer, treatment. |
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