The Mechanism Of Parthenolide Induced RIP3 Independent Necroptosis Of Colorectal Cancer Cells

Background and ObjectiveColorectal cancer (CRC) is one of the malignant tumors. The incidence and mortality of CRC in malignant tumors are 3 and 4. New cases and people die of CRC each year are more than 1.0 million and 0.55 million worldwide, showing an upward trend. To deal with CRC, surgery is the main therapeutically approach of choice. However, CRC in early stage has no obvious symptoms and most patients diagnosed CRC are in advanced stage with metastasis and thus lose opportunities of surgical operation and radiotherapy. So, the chemotherapy is irreplaceable in the treatment of malignant tumors. Unfortunately, available chemotherapeutic options for CRC are also not very effective mainly due to the occurrence of drug resistance. Most conventional anticancer agents are apoptosis inducers, regardless of their targets and mechanism. CRC cells are usually sensitive to apoptotic induction initially, but become resistant eventually through dysregulation of apoptotic machinery. Accordingly, to improve the overall survival of patients with CRC, there is an urgent need to develop effective treatment for CRC, especially with novel cell death type that different from apoptosis.Parthenolide (PTN), which is the major sesquiterpene lactone isolated from extracts of Mexican Indian medicinal plants such as feverfew, has been used conventionally to treat migraines, rheumatoid arthritis and inflammation for several centuries. Its formula is C15H20O3 and the relative molecular weight is 248.32. Recently it has been reported that PTN played a role on antitumor in vitro, for instance hepatoma, cholangiocarcinoma and primary acute/chronic myeloid leukemia. In addition, PTN enhances sensitivity of resistant cancer cell to other antitumor agents. Previous studies showed that PTN inhibited cancer through proliferation inhibition, apoptosis induction and necrosis of cancer cell lines. However, the effects and the molecular mechanism of PTN against colorectal cancer remain unclear. In view of this, the effects and its mechanism of PTN against colorectal cancer was explored in our study.Cell death has been subdivided into apoptosis and necrosis morphologically. Apoptosis is the process of programmed cell death, while necrosis is a form of traumatic cell death that occurs under a variety of tremendous physiological and pathological conditions. Recent studies have found that some death like apoptosis was launched by a specific factor, according to certain pathways and the procedure. The controlled necrosis was named Necroptosis. Necroptosis makes it possible that necrosis can be controlled and makes a revolutionary impact on scientific research and clinical treatment of diseases. Recent studies have also found that necroptosis can get rid of multi-drug resistance of the clinical antitumor medicine, improving the clinical curative effect.Receptor-interacting protein 3 (RIP3) mediated necroptosis is one of the classical pathways and RIP3 plays a key role in necroptosis induced by tumor necrosis factor α (TNF-α) and RIP3 is a molecular switch between apoptosis and necrosis in TNF-α treated NIH-3T3 cells. RIP3 does not affect RIP 1-mediated apoptosis, but is required for RIP1-mediated necrosis:RIP1 is phosphorylated by RIP3 and then form complex Ⅱ containing RIP1 and RIP3 that stimulates other enzyme to induce necroptosis. RIP3 is further enhanced by pan-caspase inhibitor z-VAD-FMK and also responsible for necrosis in other cells. Zhang and his colleagues have shown that necroptosis has different mechanisms including RIP3-dependent and RIP3-independent pathway. So we use two isogenic cell lines, mouse embryonic fibroblast (MEF) and NIH-3T3 that are either proficient or deficient in RIP3 to explore the mechanism of necroptosis induced by PTN.Methods and content:The study was divided into five parts.1 Evaluation the effect of PTN on the growth of colorectal cancerAccording to our established method, an orthotopic visualization model of CRC was adopted. Situ solid tumors were observed in the imaging system after inoculating for 12 days and then nude mice with colorectal cancer were randomly allocated to Vehicle controls and PTN (10 mg/kg/day) group. Each group contained six animals. Nude mice weight was recorded and fluorescence intensity of the tumor in vivo was detected under imaging system during the experiment.2. The effect of PTN on SW480 cell in vitroAfter treated with different concentrations of PTN (5 μM,10 μM,20μM and 40 μM), SW480 cells were cultured for 12 hours and 24 hours, respectively. MTT assay was used to detect the cell viability.3 Verification cell death of PTN on SW480After treating with PTN (20 μM), SW480 cells were cultured for different time, respectively. ELISA test and Western blot were used to analysis the effect of PTN on Caspase-3. The effect of z-VAD-FMK on SW480 cell activity with PTN was detected by MTT assay. Flow cytometry was used to detected apoptosis peak. Hoechst33342/ PI staining method was used to observe cell morphology and calculate the apoptotic and necroptotic rates of SW480. Flow cytometry was used to detect the effect of apoptosis and necrosis the content of reactive oxygen species (ROS) and the change of mitochondrial membrane potential (MMP).on SW480 cell with PTN.4 Regulating mechanism of PTN inducing SW480 necroptosis.Two isogenic cell lines, MEF (RIPS-/- and RIP3+/+) and NIH 3T3 derived A and N cell that are either proficient or deficient in RIP3 were used to explore the mechanism of necroptosis induced by PTN. After MEF (RIP3-/- and RIP3+/+) and NIH-3T3 derived A and N cell treated with different concentrations of PTN, cell were cultured for 12 h and 24 h. Then MTT assay was used to detected the effect of PTN on NIH 3T3 (A and N) and MEF (RIP3-/- and RIP3+/+) cell activity. Then Western blot was used to detect its mechanism.5 Identification cell death of PTN on A cellAfter treating with PTN (20μM), cells were cultured for 12 hours. Hoechst33342/PI staining method was used to observe cell morphology. Flow cytometry was used to detect the effect of apoptosis and necrosis. TEM was used to observe cell ultrastructure.Statistical MethodsSPSS 13.0 statistical soft was used. Measurement data was expressed as x±Sd. Comparisons between groups were performed using one-way ANOVA or independent samples t-test and multiple factors were performed using two repeated measurement factor analysis of variance. P<0.05 indicates statistical significance.Results1 PTN inhibited the growth a of colorectal cancer in Nude MiceIn vivo, PTN significantly decreased the relative fluorescence intensity of tumor compared to that of control. PTN can inhibit the growth of colorectal cancer in nude mice.2 PTN reduced cell viability of SW480MMT assay showed that PTN reduced cell viability of SW480 significantly by a time-dependent and dose-dependent manner. Cell viability was close to 50% with PTN 20μM, so 20μM was chose for subsequent experiment.3 PTN induced SW480 necroptosisApoptosis has been reviewed as caspase-mediated cell death. In our study, after SW480 cells treated with PTN, Caspase-3 activity could not improve significantly and the death was not prevented by the Pan-caspase inhibitor z-VAD-FMK. In addition, flow cytometry could not detect the apoptosis peak. Hoechst33342 staining showed dying cells treated with PTN did not exhibit apoptotic characteristics including apoptotic nuclear fragmentation and apoptotic body. Cells permeable to PI increased proportionally to the increment of incubation time, indicating the loss of plasma membrane integrity which was necroptotic characteristics. Then we found that cell death of SW480 inducing by PTN mainly was necrosis (PI staining positive) rather than apoptosis (Annexin V-FICT positive), by staining PI and Annexin V-FICT, measured by flow cytometry. Necroptosis was accompanied by a MMP dissipation. This MMP dissipation was possibly resulted from inner mitochondrial membrane (IM) permeabilization caused by a large amount of ROS.When three SW480 cells was treated with PTN, ROS of cell was increased significantly and MMP of cell decreased significantly.4 PTN inducing SW480 necroptosis was COX-2-dependent but not RIP3-dependent.We first inferred that PTN induced N cell (RIP3+/+) necroptosis was RIP3 dependant. However, the results showed that, PTN induced A cell (RIP3-/-) death but not N cell (RIP3+/+) and cell viability was no statistical significance between MEF (RIP3-/- and RIP3+/+) which indicated that PTN inducing cell necroptosis was RIP3-independent. We then wanted to know the mechanism underlying the difference of PTN on NIH-3T3 and MEF. PTN was specific inhibitor of NF-κB and NF-κB could regulate the expression of COX-2. In addition, expression of COX-2 was positive on 80% CRC. We inferred that PTN induced cell necroptosis was COX-2 relative. Our results showed that the expression of COX-2 was positive on A cell but negative on N cell and there were both positive on MEF (RIP3-/- and RIP3+/+) cell, indicating that COX-2 played a role on necroptosis induced by PTN. When three cells (MEF, NIH-3T3 and SW480) were treated with PTN, the expression of COX-2 decreased. The results demonstrated that PTN-induced necroptosis was COX-2-dependent.5 PTN induced A cell (RIP3-/-) necroptosisHoechst33342 staining showed dying cells did not exhibit apoptotic characteristics including apoptotic nuclear fragmentation and apoptotic body. Cells permeable to PI increased proportionally to the increment of incubation time, indicating the loss of plasma membrane integrity which was necroptotic characteristics. Then we found that cell death of SW480 inducing by PTN mainly was necrosis (PI staining positive) rather than apoptosis (Annexin V-FICT positive) by staining PI and Annexin V-FICT, measured by flow cytometry Cellular nucleus were swelling and endoplasmic reticulum went expansion and there were an extensive dilation of mitochondrion and formation of autophagosomes but no significant apoptotic body on A cell treated with PTN. The results demonstrated that PTN induced A cell (RIP3-/-) necroptosis.Conclusions1 PTN inhibited the growth of orthotropic CRC in BARB/c nude mice in vivo.2 PTN induced necroptosis both in SW480 cell and NIH3T3 derived A cell.3 The necroptosis was COX-2-dependent but independent of RIP3 pathway.