| According to the report of global cancer statistics 2018,lung cancer remains the leading cause of new cases and cancer deaths.The treatment of lung cancer is a combination of surgery,chemotherapy,radiotherapy and immunotherapy.However,chemotherapy is still an important treatment for patients with cancer recurrence and distant metastasis.Postoperative treatment with chemotherapy could prolong the survival of patients.However,chemotherapy has serious side effects with poor targeting,repressing the immune system and reducing the quality of life of patients.Therefore,to improve the selectivity and reduce the toxicity of drugs,alleviate the inhibition of drugs on immune system,increase the efficacy of drugs have been paid much attention in cancer drug treatment.In addition to inducing apoptosis of tumor cells,radiotherapy and chemotherapy treatments are inevitable to produce senescent tumor cells.The characteristics of senescent tumor cells are as follows:1)cell cycle arrest,enlarged and flat cell morphology,nuclear enlargement or polynuclear formation;2)active metabolism,such as increased activity of lysosomal ? galactosidase(Senescence-associated-?galactosidase,SA-?-gal),which can be used as an unique marker of cell senescence;3)up-regulated expression of DNA damage marker y-H2AX and transcription factor NF?B.In addition,the most important feature of senescence is senescence-associated secretory phenotype(SASP),which contains numerous of proteins and enzymes,such as cytokines(such as IL-1,IL-6,IL-8,MCP1),growth factors(such as VEGF),and proteases(such as MMP).Many studies have confirmed that low-dose chemotherapy drugs could inhibit tumor growth via inducing senescence of cancer cells with reduced side effects.However the paracrine effects of SASP could affect the tumor microenvironment,promote the malignant transformation of adjacent cells and the malignant evolution of tumor.The senescence of tumor cells are two-faced.Therefore,inducing cancer cellular senescence on tumor growth inhibition with minimizing the side effects of SASP is a promising strategy in cancer treatment.The previous data of our lab showed that exogenous small-molecule drugs(such as docetaxel)and endogenous gene expression(such as endoplasmic reticulum chaperone protein AGR2,RCN1)could induce the senescence of tumor cells.However the effect of the conditioned-medium on the proliferation of other cells is different.It means that the morphology phenotype of senescence cells is similar,wheras the secretion phenotype of senescent cells is different.In this study,first-line chemotherapy agent doxorubicin(DOX),natural product marchantin C(Mar-C)and the endoplasmic reticulum protein RCN1(reticulalbin-1)were used as the stimuli on the induction of senescence in lung cancer cells.Firstly,we analyzed whether the different inducing stimuli of cellular senescence affected the composition and expression of SASP and produced different SASP phenotypes.Secondly,exploring small molecular compounds that could reduce or inhibit SASP,and providing effective and intelligent strategies for anti-tumor candidate drug screening and cancer therapy.Part I Analysis of cellular senescence and secretory phenotype induced by Mar-C and RCN11 Effects of Mar-C on cell senescence and secretory phenotypeThousands of natural products have been screened for their anti-tumor and antiinflammatory activities in our previous research.Among which the marchantin compounds isolated and purified from bryophytes have unique structures with antitumor activity and low toxicity.Due to the anti-tumor and anti-inflammatory activity of Mar-C at high concentration(10 ?M),which could promote the apoptosis and paraptosis of tumor cells by inhibiting microtubules,and significantly inhibit the expression of IL6 in endothelial cells and tumor cells(unpublished data).We selected Mar-C as a potential compound,and analyzed whether Mar-C could induce the senescence of tumor cells,and affect the SASP of senescent cells.Since DOX has been proved to be able to induce cell senescence,the effects of small molecular compounds on cell senescence and SASP were studied with DOX as a positive control.1.1 Low dose of Mar-C promotes the senescence of cancer cells.(1)Low dose Mar-C(2 ?M)inhibited the proliferation of lung cancer cells,but had no effect on normal cells.The results of cell proliferation showed that 2 ?M Mar-C selectively inhibited the proliferation of lung cancer cells(A549 and H1299)and inhibited the formation of cell clones.The results of MTT and EdU showed that Mar-C did not affect the growth of normal human skin fibroblasts(NHF),human bronchial epithelial cells(HBE)and human embryonic lung fibroblasts(IMR90).(2)Mar-C promotes the senescence of tumor cells.A549 cells and H299 cells showed the typical characteristics of cell senescence after 2?M Mar-C treatment for 5 days.The cells were enlarged and flat,and SA-gal staining was positive.Similarly,2 ?M Mar-C has no senescent effect on normal NHF cells.In the detection of apoptosis marker PARP,2 ?M Mar-C did not increase PARP splicing band after 1 d and 5 d in A549 cells,indicating that Mar-C mainly promoted the senescence rather than apoptosis of lung cancer cells.We found that 0.3 ?M DOX significantly induced the senescence of both tumor cells and normal cells.1.2 Mar-C-induced SASP had little effect on the proliferation of other CellsIn order to determine the effects of Mar-C and DOX on SASP,we used Mar-C-induced or DOX-induced senescent conditioned-medium to analyze their effects on cell proliferation.Mar-C-induced conditioned-medium could promote the proliferation of A549 cells,but its effect was weaker than that of DOX-induced conditioned medium.More importantly,Mar-C-induced conditioned-medium has no effect on HBE cells.The conditioned-medium induced by DOX significantly promoted the proliferation of HBE cells.Therefore,Mar-C induced SASP had a weaker proliferative effect than DOX,and had no significant effect on normal cells.2.Effects of RCN1 on cell senescence and secretory phenotypeThe endoplasmic reticulum protein RCN1,a member of calcium-binding protein CREC(Cab45/reticulocalbin/ERC45/calumenin)family,is widely expressed in normal tissues.RCN1 is highly expressed in various cancer and associated with drug resistance,invasion and metastasis.Our previous study found that down-regulation of RCN1 induces senescence and apoptosis(about 40%)in prostate cancer cells.In this research we are focuse on the senescence induced by RCN1.2.1 Down-regulation of RCN1 induces senescence of tumor cells and normal cells Down-regulation of RCN 1 expression resulted in senescence-like phenotype and S A?-gal positive staining in many different types of tumor cells,and down-regulation of RCN1 expression also promoted the senescence of normal fibroblasts.The results of cell clone formation experiment showed that down-regulation of RCN1 led to proliferative ability of tumor cells and normal cells decreased significantly.2.2 Down-regulation of RCN1 significantly induced SASP phenotypeWe used the conditioned-medium of senescent cells induced by down-regulating RCN1 for preliminary secretory phenotype analysis and found that the conditioned-medium of senescent cells could promote the proliferation of A549 cells and HBE cells.Further study showed that the senescence of A549 cells induced by Mar-C did not decreased the expression of RCN1.Therefore it is speculated that RCN1 was not involved in the senescence of A549 cells induced by Mar-C.In conclusion,Mar-C,DOX and down-regulation of RCN1 could induce the senescence of tumor cells,but their effects on the secretory phenotype of senescent cells are different.Mar-C has good selectivity for cancer cells,while DOX and RCN1 also have effects on normal cells.These results indicate that they have different mechanisms in inducing cell senescence and related senescence phenotypes(SASP).Therefore,we will use Mar-C at 2 ?M to study the mechanism of Mar-C on cell senescence and secretory phenotype in vitro.Part 2 Mechanism of cellular senescence and secretory phenotype induced by Mar-CWe further explored the mechanism of Mar-C-induced senescence,the expression and regulation of SASP.Considering the low cytotoxicity of Mar-C,the antitumor activity of low dose Mar-C was evaluated in vivo.1 Mar-C regulated the transcriptional level of SASP1.1 The expression of SASP induced by Mar-C was lower than that DOX-induced.Antibody array were used to evaluate Mar-C and DOX on inducing SASP factor secretion.Compared with the control group,Mar-C treatment increased the secretion of IL-1,IL-6 and IFN-y,but did not affect IL-8,MCP-1 and IL-10,and significantly inhibited the secretion of TNF-and IL-13.While DOX increased the expression of IL1,IL-6,IFN-y and IL-8,but had no significant effect on MCP-1.DOX decreased the expression of IL-13 and IL-10.Therefore,both DOX and Mar-C could induce expression of SASP in senescent cells,but the level of induction wass different,and the increased pro-inflammatory factors and decreased anti-inflammatory factors of DOX was stronger than that of Mar-CQ-PCR was used to determine whether DOX and Mar-C modulate the expression of related factors in SASP at transcriptional level,and then affect the secretion level of SASP.The results showed that Mar-C and DOX could regulate the expression of the major components of SASP at the transcriptional level,and the regulatory trend was consistent with the secreted proteins of SASP,but Mar-C had less effect on the mRNA expression of SASP than DOX.1.2 Mar-C regulated expression of SASP via activating NF-?BSince both Mar-C and DOX regulate the expression of SASP at transcriptional level,transcription factors may be involved in the regulation of SASP.The results of Q-PCR showed that Mar-C had no significant effect on the mRNA expression of STAT1,STAT3,Gata4 and C/EBP?.Western blotting showed that Mar-C had no effect on the transcription factors of TFE3,TFEB,STAT1 and STAT3.As the most important inflammatory regulator,we found that Mar-C significantly increased the phosphorylation level of the NF-B subunit p65 and promoted its nuclear translocalization.Further analysis showed that Mar-C could activate I?B? by upregulating the phosphorylation of IKK?/?,and activate NF-?B into the nucleus.Downregulation of p65 could significantly inhibit the expression of IL-6,IL-8,TNF-?,IL-5 and IL-10,and partially reverse the regulatory effect of Mar-C on IL-l?,IL-1? and IL4.Therefore,NF-?B is partly involved in the regulation of Mar-C on SASP,but there are still other regulation mechanisms.2 Mar-C induced cell senescence through DNA damageBoth Mar-C and DOX could induce senescence of tumor cells,but they have different effects on SASP.Since the mechanism of DOX inducing cancer cell senescence has been studied,Mar-C has little effect on proinflammatory SASP,which may be a potential anti-tumor compound.Therefore,we focus on Mar-C-induced senescence of lung cancer cells to further explore the mechanism.2.1 Mar-C induced the cell cycle arrest at GO/Glphase of senescent cells.Cell cycle arrest is an important feature of cellular senescence.We examined the cell cycle arrest of A549 cells treated with 2 ?M Mar-C.The results showed that Mar-C could inhibit the expression of CyclinD1 and arrest the cell cycle at G0/G1 phase.2.2 Mar-C induced cellular senescence partly depent on the expression of p21.p53-p21 and p16-Rb are the key signals of cell cycle arrest and senescence.Western blotting showed that Mar-C upregulated the expression of p53 and p21,and downregulated the expression of Rb and p-Rb in A549 cells.Further analysis showed that down-regulating expression of p53 or blocking activity of p53 by inhibitors did not significantly reverse Mar-C induced cellular Senescence.In addition Mar-C also significantly induced p53 mutant cell lines(H1299 and H446)senescence Therefore,the senescence of lung cancer cells induced by Mar-C is p53 independent.However,knocking down the expression of p21 could partially reverse the inhibition of proliferation of A549 cells induced by Mar-C,suggesting that the senescence of A549 cells induced by Mar-C is p53 independent and partly dependent on its up-regulation of p21 in A549 cells.2.3 Mar-C induced cell senescence through DNA damage(1)The effect of Mar-C on DNA damage and repair proteins.Because DNA damage mechanism is usually the main mechanism of drug-induced cellular senescence,we first analyzed the effect of Mar-C on DNA damage.The results of Western blotting,immunofluorescence and comet assay showed that Mar-C increased the expression of y-H2AX in A549 cells in a time-dependent manner,and there was clear foci of y-H2AX in the nucleus.In addition,Mar-C could down-regulate the expression of BRCA1 phosphorylation and RAD51 in A549 cells,and inhibit the mRNA expression of various DNA damage repair related genes.(2)Mar-C induced DNA damage through ROS.Based on the rapid DNA damage(30 min)induced by Mar-C and the phenolic hydroxyl in its chemical structure,it is inferred that ROS is the cause of rapid DNA damage.The results showed that ROS in A549 cells treated with 2 ?M Mar-C increased significantly,which was consistent with the time line of DNA damage.Antioxidant N-acetyl-Lcysteine(NAC)could reverse the DNA damage induced by Mar-C,suggesting that Mar-C could induce DNA damage by up-regulating the level of ROS production.3 Mar-C significantly inhibited the growth of tumor and prolonged the survival time of mice.According to the effect of Mar-C on cellular senescence,we further analyzed whether Mar-C could exert anti-tumor activity by inducing lung cancer cell senescence in vivo.Two animal models,the xenograft model with A549-luc cells(luciferase-expressing A549)and the homograft mode with LLC cells,were constructed.3.1 Mar-C inhibits the growth of tumor via inducing the senescence of cancer cells The results of in vivo imaging experiments showed that the fluorescence intensity and average tumor volume decreased significantly after Mar-C treatment in nude mice,indicating that Mar-C could inhibit tumor growth.The senescence staining of frozen sections and immunohistochemical staining of Ki-67 showed that Mar-C could significantly promote the cellular senescence and inhibit the proliferation activity of tumor cells.3.2 Mar-C had little side effects and prolonged the survival time of tumor-bearing miceBy analyzing the weight change and liver function evaluation of the nude mice in each group,it was found that the bodyweight of the nude mice in DOX group decreased significantly,but there was no difference between Mar-C group and the vehicle group on the bodyweight of mice.In addition,DOX had significant hepatotoxicity,while MarC had no effect on liver function in nude mice,suggesting that Mar-C was less hepatotoxic and did not lead to liver function damage in mice.Compared with DOX,Mar-C could prolong the survival time of tumor-bearing mice.3.3 Mar-C in combination with immunomodulator CS exerts synergistic effectSince Mar-C has no significant effect on pro-inflammatory SASP,it may not significantly inhibit the immune response.The anti-tumor effect of Mar-C in combination with curdlan sulfate(CS),an immunomodulator,was investigated.A mouse model of LLC cells was established to evaluate the efficacy of the combined therapy.The results showed that DOX group,Mar-C group and Mar-C combined CS group had significant inhibitory effect on tumor growth,and the combination of Mar-C and CS showed synergistic effect.The results of Ki-67 immunohistochemical staining showed that Ki-67 staining in Mar-C group and combined group was weaker and showed a good inhibitory effect on the proliferation of tumor cells.Moreover,the movement ability of DOX group was weakened and the body weight of DOX group was decreased significantly,while that of Mar-C group was not changed.The body weight of Mar-C in combination with CS group was increased.The spleen nndex showed that DOX had a potential immunosuppressive effect on the spleen,while Mar-C and CS combined group had an increased spleen index with no immunosuppressive effect.There was no significant difference in liver function between Mar-C Group and vehicle group.Based on the above results,Mar-C has antitumor activity and no significant toxic and side effects by inducing the cancer cell senescence and inhibiting the proliferation of tumor cells.Part 3 Mechanism of RCN1 on regulating the cellular senescence and secretory phenotypePrevious data showed that down-regulation of RCN1 could induce the senescence of many kinds of tumor cells and normal cells without cell selectivity,and the conditioned medium of senescent cells significantly promoted the proliferation of other cells We explored the mechanism of RCN1-induced cell senescence and regulation of SASP.1 Down-regulation of RCN1 promotes SASP expression at transcriptional level1.1 Down-regulation of RCN1 promoted the secretion of inflammatory factors of SASPThe results of protein array showed that down-regulating RCN1 led to IL-1,IL-6 and TNF-? increased significantly,IL-8 and IFN-y also increased,while MCP-1,IL-10 and IL-13 decreased.The changes of IL-6 and IL-1 were the most significant factors in SASP.Although the relative content of anti-inflammatory factors was lower,the secretion still decreased.Therefore,down-regulation of RCN1 could influence the expression of SASP at the translation level.1.2 Down-regulation of RCN1 promoted transcription of inflammatory SASP factorTo determine whether the down-regulation of RCN1 induces changes in SASP secretion due to expression of mRNA level.Q-PCR was used to analyze the mRNA changes of SASP related factors after down-regulation of RCN1.The results showed that the expressions of IL-6,IL-8,IL-1?,TNF-?,ICAM and COX2 were significantly increased after RCN1 was down-regulated,while IL-1? was down-regulated.1.3 NF-?B and STAT1 may be involved in the transcriptional regulation of SASP by RCN1The mRNA expression was regulated by transcription factors.Western blotting showed that down-regulating RCN1 expression inhibited the expression and activation of transcription factor STAT3.However,down-regulating RCN1 could promote the phosphorylation of transcription factors p65 and STAT1,Thus we speculate that p65 and STAT1 may be involved in the regulation of SASP by RCN1.2 Down-regulation of RCN1 led to DNA damage2.1 Down-regulation of RCN1 induced cell cycle arrest at S phaseThe regulation of SASP is different from that of Mar-C,therefore we study the mechanism of senescence induced by RCN 1-down-regulation.Western blotting showed that p53 and p21 were up-regulated and Rb phosphorylation was decreased in A549 cells,while down-regulated RCN1 could induce senescence of p53 mutant cell lines(H1299,U251 and PC3).Thus down-regulation of RCN1 induces cell senescence in a p53-independent manner,and the p21-Rb pathway plays an important role in downregulating RCN1-induced senescence.Further study showed that down-regulation of RCN1 significantly inhibited expression of Cyclin Ain A549 cells,and cell cycle arrest in six tumor cell lines occurred in S phase.2.2 Down Regulation of RCN1 led to DNA damage and inhibition of DNA repair S phase is the period of DNA synthesis and replication,thus we examined the effect of down-regulation of RCN 1 on DNA damage.The results exhibited that down-regulation of RCN1 inhibited the DNA synthesis,significantly increased the expression of yH2AX,accompanied by clearly foci accumulation in the nucleus.Down-regulation of RCN1 differentially regulated the mRNA level and protein level of multiple DNA repair-related factors(phosphorylation of BRCA1 and expression of RAD51 were inhibited).2.3 The localization and function of RCN1 were not limited to endoplasmic reticulumWe explored the distribution of RCN1,and found that both DNA-damaging drugs and ionizing radiation could induce the nuclear foci aggregation of RCN1,whereas nonDNA-damaging drugs did not.Furthermore,y-H2AX foci and RCN1 foci could be colocalized upon DNA damage drugs,but not in ionizing radiation-induced DNA damage.This may be related to the different types of DNA damage caused by drugs and ionizing radiation.3 Down-regulation of RCN1 affected alternative splicing,regulates DNA damage and SASP related pathwaysBased on the discovery between RCN1 and DNA replication,DNA damage repair,proteins interacting with RCN1 were collected by immune co-precipitation and identified by mass spectrometry.GO analysis revealed that the interacting protein were related to alternative splicing.Function assay showed that down-regulation of RCN1 resulted in the inhibition of skipping exon,and overexpression of RCN1 could enhanced the fluorescence intensity of the alternative splicing luciferase reporter plasmid.RNA-sequence analysis showed that down-regulation of RCN1 given rise to the significant variation of alternative splicing in 5 types.In addition,GO analysis of significant alternative splicing events were associated with DNA damage repair,replication,inflammatory response and NF-?B pathway.4 Down-regulation of RCN1 increased chemosensitivity of tumor cells4.1 Bioinformation analysis of RCN1Bioinformatics analysis showed that RCN1 was generally expressed in tissues and organs,and higher in tumor cells than in normal tissues,and was negatively correlated with patient survival.The expression of RCN1 in 5 normal cells and 9 tumor cells was detected by Western blotting,which was consistent with the bioinformatics analysis.4.2 Down-regulation of RCN1 increased the drug sensitivity of tumor cellsDrug sensitivity experiments showed that down-regulation of RCN1 could increase the sensitivity of A549 cells to DNA damage chemotherapeutic drugs and targeted chemotherapeutic drugs.Whereas overexpression of RCN1 resulted the tolerance of A549 cells to some drugs.Taken together,we speculated that RCN1 may be related to the drug resistance of cancer treatment.In this chapter,we investigated the mechanism of down-regulated RCN1 on SASP in induced senescent cells.In our research,the expression of RCN1 could affect DNA damage repair and chemotherapeutic sensitivity of cancer cells.In conclusion,RCN1 regulated DNA damage and cell senescence through alternative splicing.This could provide a new mechanism for cancer therapy research.Innovation:(1)Mar-C selectively induces the senescence of tumor cells,with low toxicity and less SASP;(2)We first reported that down-regulation of RCN1 induces cellular senescence.The induction of DNA damage and regulation of SASP induced with RCN1 are related to alternative splicing;(3)SASP is significantly different in composition and expression due to the senescenceinduced by diverse stimuli.Deficiency:(1)The regulation of Mar-C on other transcription factors of SASP has not been elucidated and needs further study;(2)The regulation mechanism of SASP induced by RCN1 is not well studied;(3)The interacting alternative splicing proteins with RCN1 are not validated in cells. |
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