The Mechanism Of Marchantin C Promotes Cell Death And Inhibits Migration And Invasion In Lung Cancer

Lung cancer is one of the most common cancers in terms of the highest incidence and mortality throughout the world. There were 1.82 million new cases and 1.56 deaths due to lung cancer globally in 2012. The highest rates are in North America, Europe and East Asia, with over a third of new cases in China. Rates in Africa and South Asia are much lower. The mortality rate of lung cancer increased by 465 percent over the past 30 years in China, which make lung cancer to be the number one cause of tumor related deaths.Lung cancer is classified into non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) according to their pathological features. Treatment on SCLC include radiotherapy and chemotherapy, while treatment on NSCLC are comprehensive treatments which contain surgery, radiotherapy and chemotherapy. Despite advances of operation method and application of new targeted drugs, it is regrettable that 5-year survival rate of lung cancer is still low. The overall 5-year survival rate of non-small cell lung cancer is only 15%. The difficult of early diagnosis is one of the most important reason of low survival rate in lung cancer. With the difficult of early diagnosis, most of the patients are diagnosed as advanced stage so that they are not suitable for surgery, as a result, effect of chemotherapy seem to be rather important. Unfortunately, NSCLC is quite intensive to classical chemotherapeutic drugs compared with SCLC. The application of EGFR targeted drugs introduced light to the chemotherapy of NSCLC, but we often realized the limitation of single-targeted drugs for the compensatory effect. Hence, it is rather necessary and urgent to explore new multi-targeted drugs for chemotherapy of NSCLC.Natural products are the treasure of resources of new drug all the time for their various structures and biological activities. Taxol and vinca alkaloids which are first-line chemotherapeutic drugs of lung cancer were isolated from yew trees and catharanthus roseus. Allicin and curcumin showed significant anti-cancer effect, too. Now we pay our attention to bisbibenzyls isolated from liverwort plants after a screen for multiple natural products. Bisbibenzyls have shown a lot of biological activities, such as anti-inflammatory effects, anti-tumor effects, anti-fungi effects, and so on. We choose Marchatin C (MC) to be our drug for research among preliminary selection of multiple bisbibenzyls. We found MC show an significant anti-cancer effect in vivo and in vitro. Furthermore, we explored the mechanisms of cell death and inhibition of invasion and migration induced by MC. We indentified MC as a novel chemotherapeutic drug of lung cancer.Part I Marchantin C induces apoptosis in lung cancer cells by ROS mediated DNA damageBisbibenzyls which were derived from bryophytes have draw attention from peoples for their various structures and biological activities. Riccarding D and Marchantin M have been reported to show a siginificant anti-tumor effect on a lot of cancers via muiltiple death pathways such as apoptosis and autophagy. Now we focused on the anti-tumor effect of Marchantin C and explored mechanisms of cell death induced by MC.1>, MC showes significant antitumor effect in vitro and in vivo.1. To assess the inhibitory effect of MC on lung cancer cell lines (A549, H1299, H292, H460), MTT assay was firstly performed. MC led to a remarkable viability inhibition of all four lung cancer celllines with an IC50 about 15μM.2. To study whether MC inhibit the growth of lung cancer cells in vivo, a xenograft model was used. We found tumors obtained from mice injected by MC show an obvious reduction on both volume and weight without remarkable differences on body weight. Interestingly, MC didn’t s show an remarkable myelotoxicity and lier disorders like classical chemotherapeutic drugs, which means less side reaction and more comfort on patient.2、MC induces apoptosis in lung cancer cells1. We found that MC induce activation of caspase-3 and cleaveage of PARP which are key molecules in apoptosis pathway. PARP was observed to be cleavaged after 12h of MC treatment which revealed that apoptosis was not an early stage event in MC-induced cell death.2. Microarray analysis indicated that MC may induce DNA damage which was a common inducement of apoptosis. In similar fashion with PARP, DNA damage was observed after 12h of MC treatment. Besides, mitochondrial damage and ROS accumulation was observed after 12h of MC treatment, too. ROS scavenger NAC showed a remarkable protection on both DNA damage and PARP cleavage, which convinced us that MC may induced apoptosis in lung cancer cells by ROS mediated DNA damage.3. Inclusion of zVAD-fmk(a broad-spectrum caspase-inhibitor) with MC show an obviously protection on cell death, but not all of the cells were protected from death. Besides, cells treated by MC showed significantly cytoplasmic vacuolization which wasn’t a typical morphologic change of apoptosis. We believed that there were other death pathways involved with Mar C induced death except apoptosis.Part Ⅱ Marchantin C induces paraptosis in lung cancer by inhibition of proteasome activities and microtubule dynamicsWe found that there were other death pathways were involved in MC-induced cell death except apoptosis. We will study the role of autophagy, necroptosis and paraptosis on MC-induced cell death and explore the mechanisms of them.1、Neither autophagy nor necroptois is involved in MC-induced cell vacuolated death1. Firstly, we detected expression level of autophagy-related proteins. We found that LC3, the most famous marker of autophagy, was persistently up-regulated in lung cancer cells treated by MC. Since MC notedly inhibited AKT-mTOR pathway which was an famous negatively-regulatory pathway of autophagy, p62 which was always degraded via autophagy didn’t show an obvious reduction. To find whether MC induced cell death was involved with autophagy, we tested the effect of autophagy inhibitors on vacuolization and cell death. Inclusion of 3- Methyladenine (3-MA), chloroquine (CQ) and E-64D couldn’t protect cell from death and vacuolization on lung cancer cells. Moreover, siRNA of Atg 5 didn’t show a protectionh, either.2. We also tested the effect of necroptosis inhibitor, necrostatin couldn’t protect cell from cell death and vacuolization, either. These results convinced us that Neither autophagy nor necroptois is involved in MC-induced cell vacuolated death.2 Marchantin C inhibits the activities of the proteasome and triggering ER stress1. We used cell organelles’ trackers to find the source of vacuole membrane. ER-Tracker Red showed an obviously vacuole image under MC treatment by confocal laser microscopy. Besides, we could observe swelling and vacuolization of ER by electron microscopy.2. ER stress was one of the common reasons of ER swelling. We detected several molecular markers of ER stress. GRP78 increased after a short exposure to MC and persistently sustained at a high level in lung cancer cells. eIF2a phosphorylation was upregulated in response to MC atlh and peaked at 12h, which inhibits global proein synthesis while activating translation of ATF4. Act D and cycloheximide which can block global proein synthesis and relieve ER stress show a remarkable protection on MC-induced cytoplasmic vacuolization and cell death. These results convinced us that ER stress played an important role in MC-induced cell death.1、Proteasome dysfunction was one of the common reasons of ER stress. We found that MC showed a significantly inhibition on trypsin-like activity of proteasome without any effects on expression of proteasome subunits. Hence, MC and MG132(another proteasome inhibitor) showed an obviously synergistic action on both cell death and vacuolization, which convinced us that proteasome dysfunction mediated ER stress was necessary on MC-induced vacuolated cell death. Extensive cytoplasmic vacuolization originate from ER, casepase-independent cell death, blocked by CHX and Act D, all the results suggested that MC induced cell death was similar to paraptosis.3、The role of MAPKs in MC-induced paraptosisSince it has been reported that P38, ERK and JNK pathways were involved with paraptosis. We detected the role of these pathways in MC induced paraptosis. ERK pathway inhibitor PD98059 didn’t show any effect on both vacuolization and cell death; JNK pathway inhibitor SP600125 showed a remarkable synergistic reaction with MC on cell death but no effect on cell vacuolization; P38 pathway inhibitor SB203580 showed an protection on MC-induced cell death but no effect on cytoplasmic vacuolization, either. Taken together, it appears that JNK was an protective pathway on MC-induced cell death, and P38 was involved with other death pathways except paraptopsis, such as apoptosis.A 11 the three MAPKs were not involved in MC-induced paraptosis.4、The role of LC3 in MC-induced paraptosisUp-regulaion of LC3 without activation of autophagy was another characteristic of paraptosis. With a lot of experiments, we convinced that persistent ER stress induced by MC was the main cause of LC3 up-regulation. Knock down of LC3 show a significant protection on MC induced paraptosis, which indicated that LC3 played an important role in MC-induced paraptosis. We further found that a part of LC3 were located in membrane of MC-induced vacuos and hyperacetylation of LC3 induced by MC may be the major reason to the failure of autophagy.5、The role of microtubule dynamics in MC-induced paraptosisTransfection of mRFP-GFP-LC3 adenovirus suggested that vacuoles induced by MC didn’t be transported to lysosome for degradation successfully. Microarray analysis indicated that MC inhibited expression of tubulins, which convinced by western blot and immunofluorescence techniques. We suggested that disorders of microtubule dynamics may obstructed vacuoles’transportation which cause accumulation and fuse of these vacuoles, and the process finally triggered paraptosis. To further convince the conclusion, we detect the combination effect of MG132 and vincristine, a famous anti-microtubule drug. Since MG132 couldn’t induce visible vacuoles under the light microscope, combination with VCR cause obviously cytoplamic vacuolization just like MC, which suggested anti-microtubule agents seemd to be a sensitizer of paraptosis.Part III MC inhibits migration and invasion in lung cancer via modulating RGS4 expressionWe have explored the mechanisms of MC-induced cell death in Part I and Part Ⅱ. As is known to all, migration and invasion is another biological characteristic of tumor except for uncontrolled proliferation. We will study the effect of MCon migration and invasion and try to explore the mechanisms of it.1、MC profoundly inhibited the invasion and migration of A549Since we have identified the mechanism of MC-induced cell death, we further study the effect of MC on migration and invasion of lung cancer cells. Wound-healing test and transwell assay indicated that MC profoundly inhibited the migration and invasion of A549.2、MC up-regulated the expression level of RGS41. Since CXCR4 and EGFR were the main membrane receptor involved with migration and invasion in lung cancer, we firstly detected the effect of MC on CXCR4 and EGFR expression. Disappointingly, MC showed no effect on CXCR4 and EGFR expression.2. We further detected the expression of RGS4 in human normal cell lines BEAS2B and HFL1 as well as NSCLC cell lines SK-MES-1, A549, H292 and H1975. In agreement with clinical results, RGS4 was negatively expressed in NSCLC cell lines. By contrast, notable up-regulation of RGS4 was observed in normal cell lines.3. We choosed RGS4 low-expressed A549 to continue our research. Since MC showed no effect on mRNA level of RGS4, protein level of RGS4 was remarkablely up-regulated in A549 cells by MC.3、Over-expression of RGS4 in A549 cells resulted in decreased invasion and migration due to inhibition of MMP2/9 and reversal of EMT.1. Transwell assay indicated that cells transfected with RGS4-expression vector displayed significant reduction in the number of cells invaded into the lower chamber, suggesting RGS4 impaired the capability of invasion in NSCLC cells. Wound healing assay demonstrated that RGS4 over-expression markedly inhibited cells migration into the wound areas compared with cells transfected with pcDNA3.1 empty vector, suggesting that RGS4 could exert the inhibition effect of migration and invasion on NSCLC cells.2. Western blot results showed that cells transfected with RGS4-expression vector exhibited lower level of MMP2 and MMP9 expression than that in cells transfected with pcDNA3.1 empty vector. Meanwhile, we observed that the expression of epithelial marker E-cadherin was up-regulated and the mesenchymal marker vimentin was down-regulated, demonstrating a functional role for RGS4 in regulating EMT in NSCLC cells.3. Transwell assay indicated that cells transfected with RGS4-expression vector showed significantly inhibition of CXCR4 mediated migration and invasion. But EGFR mediated migration and invasion was only partly reversed by RGS4.4、MC inhibits NSCLC migration and invasion through modulating RGS4 expressionTranswell and wound healing assay indicated that the silence of RGS4 could significantly reversed MC-induced inhibition on migration and invasion. Western blot assay showed that the silence of RGS4 could significantly reversed inhibition of MMP2/9 and reversal of EMT induced by MC, too. All the results convinced us that MC inhibits NSCLC migration and invasion through modulating RGS4 expression.Part IV Correlation analysis of RGS4 expression and its prognostic significance in NSCLC patientsWe found that RGS4 was down-regulated in NSCLC cell lines compared with normal cell lines. Further studies showed that RGS4 expression was correlated with invasion and migration. All the results suggested that RGS4 may be served as a novel prognostic biomarker. In this part, we will detect RGS4 expression of tumor specimen and explore the correlation between RGS4 expression and prognosis.1、RGS4 expression is down-regulated in NSCLC specimensClinical samples obtained from 121 NSCLC specimens and 67 normal lung tissues were collected to detect the expression of RGS4 protein by immunohistochemistry staining. According to our scoring criteria, RGS4 protein was highly expressed in 41.3%(50/121) of tumor specimens and 64.2%(43/67) of normal lung tissues, indicating that RGS4 levels in NSCLC tumor tissues were significantly lower than normal lung tissues(chi-square test, P=0.003). To verify our suggestion, Quantitative Real-Time PCR and Western blot were carried out to measure the mRNA and protein levels of RGS4 in 15 pairs of primary tumor and adjacent normal lung tissues. The results indicated that NSCLC specimens showed decreased mRNA levels compared with their adjacent normal lung tissues.2-. Correlation of RGS4 expression with clinicopathological parametersWe collected the clinicopathological characteristics of all the 121 cases, including sex, age, histology and differentiation of tumor, TNM stage, and so on. Next, we analyzed the correlation between RGS4 expression and these factors by chi-square test. Lower RGS4 expression was significantly correlated with positive lymph node metastasis (P= 0.009) and advanced TNM stage (P= 0.008). It should also be noted that there was no statistical significance between RGS4 expression and other clinicopathological variables (P>0.05).3> Relationship between RGS4 expression and clinical prognosis1. Correlation analysis of RGS4 expression and 5-year disease-free survival in NSCLC patientsUnivariate analysis (log-rank test) demonstrated that low RGS4 expression (52% versus 80.3%, P=0.002) predicted decreased 5-year disease-free survival and multivariate Cox regression analysis indicated that low RGS4 expression (P=0.020) retained their significance so that they are independent prognostic factors for unfavorable 5-year disease-free survival.2. Correlation analysis of RGS4 expression and 5-year overall survival in NSCLC patientsSimilarly, low RGS4 expression (50% versus 74.6%,P=0.005) predicted decreased 5-year overall survival and Cox regression analysis showed low RGS4 expression (P=0.041) retained their significance as an independent prognostic factor for unfavorable 5-year overall survival.Part V Conclusions and Innovation1、Conclusions1. MC showed significant antitumor effect in vitro and in vivo.1. MC induced apoptosis in lung cancer cells by induction of ROS and DNA damage.3. MC induced paraptosis in lung cancer cells by inhibition of proteasome activities and microtubule dynamics.4. MC inhibited invasion and migration in lung cancer cells by up-regulation of RGS4.5. RGS4 was down-regulated in NSCLC and served as an independent prognostic factor of NSCLC paients.2、Innovation and defects1. We firstly reported that ROS and DNA damage were involved with MC-induced apoptosis.2. We firstly reported that MC induced caspase-independent paraptosis in lung cancer cells by inhibition of proteasome activities and microtubule dynamics.3. We firstly reported that over-expression of RGS4 in A549 cells resulted in decreased invasion and migration due to inhibition of MMP2/9 and reversal of EMT.4. We firstly reported that RGS4 may serve as an independent prognostic factor of NSCLC paients.5. The role of LC3 in MC-induced paraptosis and mechanisms of how RGS4 regulate invasion and migration need to be further investigated.