A Novel Derivative Of Tetrandrine, H1, Induces Apoptosis And Autophagy In Human Lung Cancer Cells

ObjectiveCancer has been one of major causes that harm people’s health and lower their quality of life because of its high morbidity and mortality. However, the traditional method for treatment of tumor is relatively limited, mainly including surgery, chemotherapy and radiotherapy. When cancer patients are treated with chemotherapy drugs, cancer cells will be killed or inhibited. But in the meantime, chemotherapy drugs can cause normal tissue or organs damaged and suffered from toxic effect. So, the development of specific drugs targeting tumor cells is more and more urgent.Tetrandrine (TET) is a bisbenzylisoquinoline alkaloid, which is purified from the root Stephania tetrandra S. Moore. Many researches have shown that TET has excellent anti-tumor activity. H1, a novel derivative of TET which was synthesized and optimized by Prof. Wang, has been reported to exert more potent activity of anti-tumor. This paper aims to study H1-induced apoptosis and autophagy in human non-small cell lung cancer cells and further clarify the underlying molecular mechanism.Methods1. Cancer cells culture.2. Sulfonyl rhodamine B (SRB) colorimetry to detect tumor cells proliferation.3. Western Blot experiments to test the change of the protein in cells.4. Flow cytometry to detect cell apoptosis.5. RNA interference to suppress the expression of related genes.6. Using GFP-LC3B fusion protein to trace the formation of autophagosome.ResultsA novel derivative of Tetrandrine, H1, induces apoptosis in Human NSCLC cells 1. H1concentration-dependently inhibited the proliferation of five human non-small cell lung cancer cells, including A549, Calu-1, H1792, H157and H460cells.2. H1induced the activation of caspase cascade in Calu-1, A549and H1792cells.3. H1up-regulated DR5expression and knockdown DR5protected cells from apoptosis induced by H1in Calu-1, A549and H1792cells.4. H1activated endoplasmic reticulum stress in Calu-1, A549and H1792cells thus increase the expression of CHOP.5. Knockdown CHOP inhibited the expression of DR5and H1-induced apoptosis in Calu-1, A549and H1792cells.6. H1down-regulated the level of c-FLIP protein in Calu-1, A549and H1792cells. Over-express c-FLIP will protect cells from H1-induced apoptosis.A novel derivative of Tetrandrine, H1, induces autophagy in Human NSCLC cells1. H1up-regulated MAP1LC3B-type Ⅱ in Calu-1, A549and H1792cells.2. H1increased the eGFP-MAP1LC3B puncta in Calu-1-EGFP-MAP1LC3B cells.3. Knockdown ATG5or ATG7inhibited H1-induced up-regulation of MAP1LC3B type Ⅱ in Calu-1cells.4. Knockdown ATG5or ATG7decreased the EGFP-MAP1LC3B puncta in Calu-1-EGFP-MAP1LC3B cells.5. Autophagy inhibitor, LY294002and E64d, effectively suppressed the H1-induced autophagy.6. Knockdown ATG5or ATG7promoted H1-induced apoptosis in Calu-1cells.Conclusions1. H1possessed an antitumor activity by inducing apoptosis in human non-small cell lung cancer cells. 2. H1up-regulated the expression of CHOP by activating endoplasmic reticulum stress, resulted in the up-regulation of DR5expression and H1-induced apoptosis.3. Anti-apoptotic protein c-FLIP played an important role in H1-induced apoptosis in lung cancer cells.4. H1induced autophagy in human non-small cell lung cancer cells.5. Autophagy, induced by H1, inhibited H1-inducing apoptosis in human non-small cell lung cancer cells.We found that H1played a role of anti-tumor by inducing apoptosis in human non-small cell lung cancer cells. Then we further clarified the underlying molecular mechanism of H1-induced apoptosis. H1increased the expression of CHOP, a crosstalk protein molecule between ER stress and cell apoptosis, which resulted in the up-regulation of DR5expression and H1-induced cell apoptosis. In addition, anti-apoptotic protein c-FLIP played an important role in H1-induced apoptosis in lung cancer cells.At the same time, we found that H1induced autophagy in human non-small cell lung cancer cells. Autophagy, induced by H1, inhibited H1-inducing apoptosis in human non-small cell lung cancer cells.