Antitumor Activity Of Novel Derivative Of Quinazoline WYK431and Its Mechanism

Gastric cancer (GC) is one of the most common tumors and remains the second leading cause of cancer mortality in the world. Geographically, Asian, Eastern Europe and South American countries have a higher incidence rate of GC than the United States and Western Europe.The incidence and mortality of gastric cancer accounting for approximately over400,000new cases and over300,000deaths occurred annually making it the first most common malignancy and the first leading cause of cancer death in China. Conventional treatment modalities, including surgery, radiotherapy, and chemotherapy, play a role mainly in patients with early disease. Despite the better response rates (RRs) and tolerability with various new-generation combination regimens using capecitabine, oxaliplatin, SI, docetaxel, and irinotecan [6-9], the OS (overall survival) outcome of AGC (advanced gastric cancer) patients remains dismal. With the greater understanding of the biology and under lying molecular basis of carcinogenesis, new therapeutic approaches are needed for advanced gastric cancer.Many factors have been identified to be related to carcinogenesis and progression of gastric cancer. These factors include HER-2and EGFR overexpression, PIK3CA mutations and PTEN loss. HER-2overexpression is observed in10%-38%of gastric cancer tumor samples and EGFR overexpression is observed in27%-44%of gastric cancer cases. Activation of these receptors leads to homo-or heterodimerization that in turn initiates phosphorylation cascades and subsequent activation of the PI3K-Akt-mTOR and Ras-Raf-MEK-ERK pathways, which are important in cancer cell proliferation and survival. The PI3K-Akt-mTOR pathway is frequently activated in gastric cancer. PIK3CA activating mutation was reported in4%-36%of gastric cancer cases and PTEN loss was reported in20%-36%of cases.The prevalent expression of phospho-Akt (29%-86%) and phospho-mTOR (47%-64%). This can be mediated either by the overexpression of upstream receptors or by constitutively enhanced PI3K activity caused in turn by activating mutations of PIK3CA or PTEN loss. The Ras-Raf-MEK-ERK pathway is another key signaling pathway downstream of HER. For gastric cancer, K-RAS mutation was observed in2%-20%of cases and B-RAF mutations was observed in0%-2.7%of cases. Thus far, the human epidermal growth factor receptor (HER) pathway and phosphatidylinositol-3-kinase (PI3K)-Akt-mammalian target of rapamycin pathway have emerged as potential avenues for targeted therapy in AGC patients. The promising efficacy results of the Trastuzumab for Gastric Cancer trial led to the approved use of trastuzumab-based therapy as first-line treatment for patients with HER-2+AGC. Phase III data are awaited for other targeted agents, including cetuximab, panitumumab, lapatinib, and everolimus.It is previously reported that WYK431, a novel synthetic quinazoline derivative, shows potent antiproliferation activity against a broad spectrum of human cancer lines and BGC823cell line is the most sensitive to its treatment with an IC50value of2.16μM. But possible anticancer mechanisms of WYK431remain unclear. In this study, human gastric cancer BGC-823cells was used to investigate the effects and potential mechanisms of WYK431. To evaluate the antitumor activity of WYK431in vivo, BGC823bearing BALB/c nude mice were treated with WYK431at the doses of5mg/kg,10mg/kg and20mg/kg. Our results showed that WYK431administered at5mg/kg,10mg/kg and20mg/kg of WYK431suppressed tumors at28.4%,41.9%and63.6%, respectively compared with vehicle-treated animals (p<0.05). Moreover, WYK431treatment was well tolerated, with only small effects on body weight, which was consistent with the notion that WYK431preferably targets tumor cells and thus exhibited little toxicity. Hematological analysis showed that WYK431did not significantly decrease the number of white blood corpuscles. Flow cytometry was used to analyze cell cycle. Our result showed that treatment of BGC823with WYK431induced G2/M phase arrest. DAPI staining was performed to investigate the apoptosis induction effect of WYK431. Treatment with WYK431showed features of apoptotic cells such as a brightly blue-fluorescent condensed nuclei and apoptotic bodies. To further confirm the apoptosis-induced effect by WYK431, an Annexin V-FITC apoptosis detection kit was used to analyzed the percentage of the early apoptotic cells. Our results showed that WYK431induced apoptotic death of human gastric cancer BGC823cells in a dose-dependent manner. In vivo, TUNEL-positive cells with dark green fluorescent staining in the treatment group were observed, indicating a significant increased apoptosis in the treatment group compared with control group. These results suggest that WYK431induced apoptosis of tumor cells in vivo. To further characterize WYK431-induced apoptosis and investigate which apoptotic pathway WYK431activated, we examined caspase-3, and the patterns of proteolytic processing of caspase-8and caspase-9, the apical proteases in the extrinsic and intrinsic pathways respectively. Our result showed that treatment of BGC823with WYK-431induced cells apoptosis correlated with activation of caspase-3, caspase-9, suggesting that WYK-431triggered apoptosis not through extrinsic pathway but intrinsic pathway. A key step in the intrinsic apoptotic pathway is damage of mitochondria and the release of cytochrome c to activate Apaf-1, which turns on the caspase cascade. Our results showed that the treatment of BGC823cells with WYK431led to a loss of Δψm and released cytochrome c from mitochondria into cytoplasm. Mitochondrial integrity is regulated by pro-apoptotic and anti-apoptotic members of the Bcl-2group of proteins such as Bcl-2(anti-apoptotic) and Bax (pro-apoptotic). Bcl-2protects cells from the induction of apoptosis by interacting with pro-apoptotic members of the Bcl-2group and thus blocking the release of cytochrome c from the mitochondria. Our result showed that the expression of Bax was increased in cells treated with WYK431, whereas there was no change in Bcl-2protein expression, suggesting that intrinsic pathways were involved in the apoptosis process.Both PI3K/Akt and p44/42mitogen-activated protein kinase (MAPK) pathway are known to play a critical role in cell growth and survival, and they are dysregulated in gastric cancer cells. MAPK and Akt pathway have been important molecular targets for cancer prevention and treatment in gastric cancer. Our results found a remarkable decrease in the level of phosphorylated Akt and phosphorylated-Erk, however, the levels of total Akt and Erk were unaffected by WYK431. The EGF receptor plays a crucial role in the regulation of cell proliferation, differentiation, development and oncogenesis. EGFR which is upstream of both PI3K/Akt and MAPK pathway is often dysregulated in gastric cancer and contributes to tumor progression. In this study, our result showed WYK431treatment resulted in a dose dependent decrease in the level of phosphorylated EGF receptor in BGC823cells but the total protein levels of EGF receptor did not change following treatment with WYK431. It indicated that WYK431inhibited a post-translational modification of EGFR. In conclusion, WYK431is a novel small-molecule agent which inhibits BGC823proliferation through the induction of G2/M phase arrest and subsequent apoptosis via mitochondrial apoptotic pathway. In addition, WYK431potently inhibits the growth of BGC-823cells xenografted in athymic nude mice. The antitumor activities of WYK431may associated with inhibition of PI3K/Akt and p42/44MAPK pathway. Its potential as a promising anticancer agent is required further investigation. Metastatic melanoma is the most aggressive form of skin cancer with approximately46,000annual deaths worldwide. The incidence of malignant melanoma is increasing faster than any other cancer. Treatment is dependent on the stage of the disease, and for early-stage cutaneous malignant melanoma consists of surgical excision. Patients at high risk of recurrence may benefit from adjuvant therapy with IFN-α. Until2011, the only two US FDA therapies licensed for metastatic melanoma were dacarbazine and high-dose IL-2(HD IL-2). Although cytotoxic chemotherapy has been widely used in patients with metastatic disease, treatment with dacarbazine is limited by low response rates (10-15%) and an overall survival (OS) of8months. Treatment with HD IL-2also produces low response rates (<10%) and severe multiorgan toxicity, with only a minority of carefully selected patients achieving long-term, disease-free response. Development of novel agents that selectively target and inhibit key molecular mutations that drive tumorigenesis in melanoma has improved response rates. B-RAFV600E mutation is the most common oncogenic mutation in melanoma with approximately50%of patients with cutaneous melanoma. Vemurafenib is a B-RAF inhibitor that results in inhibition of the MAPK pathway in patients whose tumors harbor a V600mutation in the B-RAF gene. Phase I and II trials showed that vemurafenib had significant improvements in OS and PFS. Ipilimumab, a monoclonal antibody targeting cytotoxic T-lymphocyte-associated antigen4(CTLA-4), is a novel immunotherapy that has demonstrated significant survival advantage in metastatic melanoma.The recent success of both vemurafenib and ipilimumab has revolutionized the standard-of-care treatment for patients with metastatic melanoma. However, a major challenge is overcomeing the acquired resistance to BRAF inhibitors such as vemurafenib in melanomas, improving the response rate of ipilimumab and exploiting new targets for clinical benefit. Acquired resistance to BRAF inhibitors has been described in both MAPK-dependent and MAPK-independent pathways from patient-derived samples. Mechanisms of MAPK-dependent resistance include secondary N-RAS mutations, B-RAF truncations, elevated expression of COT kinase, C-RAF activation and acquired MEK1mutation. MAPK-independent pathways include upregulation of receptor tyrosine kinases including PDGFRβ, IGF1R, AXL, ERBB4as well as activation of PI3K/AKT/mTOR and Stat3signaling. The combination of a B-RAF and MEK inhibitor has shown promising results in melanoma patients naive to prior anti-B-RAFtreatment. Ipilimumab is also under evaluation in the adjuvant setting.In part1study, the results that WYK431potently suppresses human gastric cancer BGC823growth have demonstrated. But the target of WYK431remains unknown. The in vitro kinase inhibition assays were performed to determine kinase inhibition profile of WYK431. WYK431inhibited PI3Kinase(p110δ/p85α) at rate of80.5%, at concentration of10μM in biochemical kinase assays. Moreover, WYK431inhibited a panel of human cancer cells proliferation in a concentration-dependent manner and human melanoma cell line A375was most sensitive to WYK431. In this study, we investigated the anticancer effects of WYK431on A375cells both in vitro and in vivo as well as potential mechanisms. Results showed that WYK431inhibited proliferation, arrested S phase related to CDK2and induced apoptosis associated with activation of caspase3and caspase9rather than caspase8on A375cells. Treatment of A375cells with WYK431could result in disruption of mitochondrial membrane potential and upregulation of Bax.A375bearing BALB/c nude mice were treated with WYK431at the doses of5mg/kg,10mg/kg and20mg/kg. Our result show that WYK431administered at5mg/kg,10mg/kg and20mg/kg of WYK431suppressed tumors at18.92%,51.18%,76.60%(p<0.05) compared with vehicle-treated animals (p<0.05). Moreover, WYK431treatment was well tolerated, with only small effects on body weight. In the treatment group, tumors exhibited reduced staining of Ki67, implying a significant reduction in proliferating cells in the tumor and showed more TUNEL-positive cells with dark green fluorescent staining, indicating a significant increased apoptosis in the treatment group when compared with the control group. Angiogenesis, the formation of new vessels from an existing vascular network, play an important role in growth and metastatic dissemination of most solid tumors in human. Tumor-associated vasculature are a hallmark of a variety of human and rodent tumors in vivo and correlated with tumor growth rate, tumor metastatic potential and poorer patient prognosis in a variety of malignancies Histological analysis showed significant reductions in intratumoral microvessel density (CD31staining) of61.5%relative to controls depending on the specific tumor xenografts.Both PI3K/Akt and p44/42mitogen-activated protein kinase (MAPK) pathway are known to play a critical role in cell growth and survival, and they are dysregulated in in melanoma. Western blot analysis showed that WYK431block PI3K/Akt and p44/42 mitogen-activated protein kinase (MAPK) signaling pathway. It is reported that Stat3is constitutively activated in a majority of human melanoma cell lines and tumorspecimens examined. In addition recent study showed that activation of Stat3induced resistance to vemurafenib in melanoma cells. Our result showed that WYK431downregulated the expression of p-Stat3,but have no change on expression of total Stat3.Collectively, WYK431is a novel small-molecule agent which inhibits A375cells proliferation induces S phase arrest and apoptosis via mitochondrial apoptotic pathway. Moreover, WYK431effectively inhibited the growth of BGC-823cells xenografted in BALB/c athymic nude mice without major side effects. Its potential as a promising anticancer agent is required for further investigation.