Studies On Antitumor Activities And Mechanisms Of Triptolide And YCH337,both Of Which Overcome Tumor Drug Resistance

Drug resistance is a major cause for tumor treatment failure, which seriously impairs life quality and reduces survival time of tumor patients. Drugs that can avoid this outcome are urgently needed. In order to develop such types of anticancer drugs, a lot of work is devoted to searching for compounds that possess potent antitumor activities and can also overcome tumor drug resistance. In this study, we investigated the anti-tumor and anti-drug resistance activities and their mechanisms of two small molecular compounds, triptolide and YCH337.Our previous work showed that triptolide could elicit a high potency and a broad spectrum of antitumor activity by inducing the degradation of Rpb1 via CDK7. In this study, we studied anti-drug resistance activity of triptolide and its mechanism. The results showed that triptolide directly killed multiple drug resistance(MDR) tumor cells with an average resistance factor of 0.77 for 3 MDR cell lines. The proliferation inhibition on 2 MDR cell lines, KB/VCR and MES-SA/DX5, was nearly 2 times more potent than that on their corresponding parental cells, with resistance factors of 0.55 and 0.54, respectively. The overexpression of drug transporters, such as P-glycoprotein(P-gp), is a major cause of MDR. However, the anti-MDR activity of triptolide was not mediated by inhibiting P-gp. Triptolide(1 μM; 90 min) did not suppress the efflux function of P-gp in KB/VCR cells. And the decrease of P-gp and MDR1 m RNA occured 36 h post the treatment with 50 n M triptolide, whereas Rpb1 began to degrade as early as 4 h. Therefore, the effect of triptolide on P-gp may be an outcome of its transcription inhibition. Cancer stem cells have been considered as another critical reason for tumor MDR. Cancer stem cells within some specific tumors may be able to self-renew and colony formation assays could be used to reflect this. The colony formation rate of MDR KB/VCR cells increased by approximately 10% when compared with that of parental KB cells. Triptolide inhibited the colony formation of KB and KB/VCR with IC50 values of 0.68 n M and 0.41 n M, respectively. Transcription factors such as c-Myc, SOX-2, OCT-4 and NANOG are critical for cancer stem cells. Triptolide reduced c-Myc protein and c-myc m RNA but only slightly lowered SOX-2, and did not change OCT-4 or NANOG in these cells. These transcription factors were not correlated with triptolide-induced cell killing. However, Rpb1, the largest subunit of RNA polymerase II, was critical in mediating triptolide’s inhibition of MDR cells. Our data showed that triptolide elicited antitumor and anti-MDR activity through a universal mechanism: by activating CDK7 by phosphorylating Thr170 in both parental and MDR cell lines and in SK-OV-3 cells. Triptolide increased the level of CDK7 phosphorylation at threonine 170(p-T170-CDK7) that activated the enzymatic activity of CDK7 and thus induced degradation of Rpb1 in both MDR and parentl cells and in SK-OV-3 cells. The CDK7 selective inhibitor BS-181 partially rescued cell killing induced by 72 h treatment of triptolide which may be due to partial rescue of Rpb1 degradation. We suggest that a precise phosphorylation site on Rpb1(Ser1878) identified by mass spectrometry analysis was phosphorylated by CDK7 in response to triptolide. In addition, we found that XPB and p44, two transcription factor TFIIH subunits did not contribute to triptolide-driven Rpb1 degradation and cell killing although XPB was reported to covalently bind to triptolide. Several clinical trials are underway to test triptolide and its analogues for treating cancer and other diseases, so our data may help expand potential clinical uses of triptolide as well as offer a compound that overcomes tumor MDR. Future investigations into the primary molecular target(s) of triptolide responsible for Rpb1 degradation may suggest novel anti-MDR target(s) for therapeutic development.Another work studied the antitumor activity and its mechanism of a novel compound YCH337 that possesses anti-MDR acitivity. Through collaboration with Professor Chun-Hao Yang’s group, we found a novel α-carboline derivative YCH337 that elicited potent antitumor activity. YCH337 was shown to be a dual inhibitor targeting both microtubule and topoisomerase II(Top2). Microtubule and Top2 are important antitumor targets and their respective inhibitors or combinations are widely used clinically for cancer therapy. There are no drugs targeting both microtubule and Top2 used in the clinic till now. YCH337 elicited proliferation inhibition on 19 different tumor cell lines with an averaged IC50 of 0.3 μM. YCH337 significantly suppressed the growth of HT-29 xenografts in nude mice. In addition, YCH337 also inhibited the proliferation of different types of drug resistant tumor cells as potently as their parental cells. YCH337 induced microtubule depolymerization by binding to the colchicine site, which led to multipolar spindle formation and mitotic arrest in He La cells. YCH337 also suppressed Top2 and caused DNA double-strand breaks(DSB), which led to the accumulation of γH2AX. The inhibition on microtubule was more potently than that of Top2. YCH337 caused notable microtubule inhibition at a relatively lower concentration for a shorter exposure than Top2 inhibition. YCH337 induced the depolymerization of microtubule at 0.1 μM for 1 h or at 1 μM for 15 min treatment, while a higher concentration(0.2 μM) or a longer time(30 min) were needed to inhibit Top2. YCH337 induced reversible mitotic arrest but persistent DNA damage at 0.2 μM; however, the mitotic arrest caused by YCH337 at higher concentrations(≥0.5 μM) was also irreversible. YCH337 induced activation of caspases 9/8/3 and apoptosis via intrinsic and extrinsic apoptotic pathways. YCH337 also decreased anti-apoptotic proteins, such as MCL-1, c IAP1 and XIAP. YCH337 behaved similarly to vincristine(VCR) but differently from etoposide(VP-16) in inducing apoptosis. The antitumor activity of VCR was weakened when combined with VP-16, indicating that inappropriate drug combinations of microtubule inhibitors and Top2 inhibitors could not potentiate their antitumor effects. Therefore, dual targeting of microtubule and Top2 provides a new strategy for solving these issues.Taken together, this study systematically demonstrates the antitumor activities and their mechanisms of two small molecular compounds, triptolide and YCH337, which may offer new perspectives for the development of new types of antitumor drugs. With increasing understanding of tumorigenesis and tumor drug resistance, we hope to rationally design new antitumor drugs that can elicit improved therapeutic effects and overcome or avoid tumor drug resistance.