Overaccumulation of p53-mediated autophagy protects against betulinic acid-induced apoptotic cell death in colorectal cancer cells

Betulinic acid (BA) exhibits cytotoxic activity against some cancer cells. However, the molecular mechanism of BA against CRC cells was little reported. Here, we proved that BA elicited CRC cells' growth inhibition and apoptosis in a dose-dependent manner. In addition, BA treatment induced autophagy via inhibiting the AKT-MTOR signaling pathway. Inhibition of autophagy by either administration of autophagic inhibitor chloroquine or siRNA-mediated knockdown of ATG5 could augment BA-induced apoptotic cell death as well as inhibition of cell proliferation. Moreover, we found that p53 was firstly activated by short exposure to BA and then was rapidly degraded via the ubiquitin-mediated degradation pathway in both wtp53 and mutp53 CRC cells. Notably, more preferential cytotoxicity of BA was obtained in mutp53 cells (IC50 values: HT29, 125 μM; SW480, 58 μM) rather than wtp53 cells (IC50 values: HCT116, 178 μM). Further experiments demonstrated that siRNA-mediated p53 knockdown attenuated BA-induced autophagy, and forced overexpression of p53 augmented BA-induced autophagy, indicating that p53-enhanced BA-induced autophagy. Moreover, BA enhanced the sensitivity of mutp53 cells to chemotherapy drugs such as 5-FU and ADR by degradation of mutp53. Overall, our study proved that BA could induce CRC cell death by inducing apoptosis and reduce the overaccumulation of BA-induced protective autophagy by degrading wtp53 and mutp53 dependent on the ubiquitin-mediated degradation pathway to achieve killer effect, suggesting that BA might serve as a novel desirable drug for mutp53 cancer therapy.

Colorectal cancer is the third most common cancer and the fourth most common cause of cancer death worldwide. 1,2 As alternative treatments, new chemotherapy agents using novel mechanisms to induce CRC cell death are needed, whereas plants are an important source for potential anticancer compounds. [3][4][5] Betulinic acid (BA), a naturally occurring pentacyclic triterpene, has shown anticancer property in several human cancers [6][7][8][9][10][11][12][13][14][15] and no effect on untransformed cells. [16][17][18] However, the mechanism of BA-induced antitumor effect has been still on debate. 12,19 In CRC cells, BA could induce apoptosis through the mitochondrial pathway, 20 inhibited cell growth of CRC cells by downregulating Sp transcription factors in SW480 and RKO cells, 21 or by suppressing NF-κB and STAT3 signaling in HT29 cells. 22 It has also been reported that BA can function as NF-κB activator in a number of other cancer cell lines, 23 suggesting that the antitumor mechanisms of BA was dependent on tumor type.
Autophagy is a catabolic process that allows cellular macromolecules to be broken down and recycled as metabolic precursors. 24 Autophagy is usually activated in cancer cells challenged with various stresses. [25][26][27][28][29] In response to chemotherapy stress, autophagy probably has four different functions including cytoprotective, cytotoxic, non-protective and cytostatic form. [30][31][32][33][34][35] BA could induce autophagy in multiple myeloma cells and glioblastoma cells, 36 whereas autophagic flux inhibition promoted apoptosis in human multiple myeloma KM3 cells. Furthermore, BA or BA analog could induce autophagy in HeLa, A549, MCF7, SW480 and HT29 cells, and autophagic salvage counterbalanced BA-induced cell death in HeLa cells. 10,37 Although studies observed that BA could induce autophagy, the role of autophagy as a cell death mechanism was not minutely addressed for BA-treated CRC cells.
The tumor suppressor p53 is implicated in a wide range of cellular processes, such as cell-cycle arrest and apoptosis. [38][39][40] p53 binds specifically to DNA and regulates transcription of target genes, such as P21, BIM, BAX, PUMA and NOXA, to trigger cell death. 41 However, mutp53 in a large fraction of human cancers could promote evasion of apoptosis, accelerate tumor progression and chemoresistance, which is defined as mutp53 gain-of-function (GOF). 40,[42][43][44][45] Pharmacological inactivation of mutp53 and effective therapy that targets mutp53 degradation have emerged as a promising strategy to improve cancer therapy. 40,44,46 In addition, p53 can either be a positive or a negative regulator of autophagy. 47,48 At basal levels, p53 is recognized as an inhibitor of autophagy through protein-protein interactions in mitochondria. 49,50 In response to stress, p53 translocated to the nucleus and promoted autophagy by trans-activating its target genes. 51,52 BA triggered an apoptosis pathway different from standard chemotherapeutic drugs because BA-induced apoptosis was independent of p53 in neuroectodermal cancer cells, 53 melanoma cells and glioblastoma cells. 54 However, no research was reported regarding whether BA induced CRC cell death independent of p53 status and histotype, and the related detailed mechanism remains unknown.
In this study, we found that BA induced a pro-survival effect of autophagy in CRC cells and p53 can further augment this autophagy. To avoid the overaccumulation of protective autophagy, BA would induce both wtp53 and mutp53 degradation, which makes BA exhibiting preferential cytotoxicity to mutp53 cells.

BA inhibits cell proliferation and induces apoptosis in CRC cells.
To determine the anticancer effect of BA in CRC cells, HCT116, SW480 and HT29 cells were treated with different concentrations of BA, and then cell proliferation was assayed with a CCK8 regent kit. We found that administration of BA dose-dependently decreased the proliferation of all three CRC cells. IC50 values of BA against CRC cells were 178 μM (HCT116), 58 μM (SW480) and 125 μM (HT29; Figure 1a). Chemotherapeutic drug-induced cell proliferation inhibition was mainly because of the initiation of apoptosis. 55 Annexin-V/PI double staining and DAPI staining were used to estimate the effect of BA on apoptosis in CRC cells. The results showed that BA induced apoptosis in a dosedependent manner (Figures 1b and c). Finally, we further detected the activation of PARP, which is a key factor of apoptosis. 45 Results showed that BA induced PARP cleavage in a dose-dependent manner (Figure 1d), indicating that BA induced CRC cells' apoptosis. In addition, p53 protein was decreased and its target gene BAX was unchanged after BA treatment (Figure 1d), suggesting that p53 may be involved in BA-induced CRC cell death in a special manner.
BA initiates autophagy in CRC cells. Autophagy has an important role in the drug treatment of cancers. 56 To investigate whether BA could initiate autophagy in CRC cells, transmission electron microscopy (TEM) was used to monitor autophagy. 24 The formation of double-membraned autophagic vacuoles was frequently observed in CRC cells treated with BA, but not in control cells (Figure 2a). In addition, we tested the formation of acidic vesicular organelle (AVOs), another major feature of autophagy 57 in three kinds of CRC cells. Results showed that the numbers of AVOs notably dose-dependently increased in these CRC cells (Figure 2b).
To further confirm whether BA initiates autophagy in CRC cells, the processing of LC3-I to its PE-conjugated LC3-II was measured by immunoblotting. As shown in Figure 2c, BA increased the conversion of LC3-I to LC3-II in a dosedependent manner, which was further supported by the   Figure S1). The distribution of endogenous LC3 in CRC cells was also analyzed by indirect immunofluorescence staining. As shown in Supplementary Figure S2A, more specific LC3 punctuates were found in BA-treated cells compared with the control group. Finally, qRT-PCR was performed to determine the mRNA expression levels of BECLIN1, ATG7, ATG12 and ATG5, which are involved in the formation of autophagosome. 58,59 Results showed that BA induced the expression of autophagy-related genes in a dosedependent manner (Supplementary Figure S2B). These results demonstrated that BA could induce autophagy in CRC cells.
BA increases the autophagy flux in CRC cells. To determine that BA could also induce autophagy flux in CRC cells, the colocalization of LC3 and LAMP1 was firstly analyzed by indirect immunofluorescence assay, which is often used as a marker for autophagy flux. 7 The results showed that the colocalization of LC3 and LAMP1 was increased in BA-treated CRC cells compared with the control group ( Figure 3a). Another commonly used method for measurement of the autophagy flux is to monitor the conversion of LC3-I to LC3-II in the presence of lysosome inhibitor. 58 We found that BA co-treatment with E-64 day/ pepstain A or chloroquine led to further accumulation of LC3-II in CRC cells (Figure 3b), indicating that BA promoted the autophagy flux in CRC cells. However, interestingly, the mRNA and protein expression of SQSTM1, another marker of the autophagy flux, which is efficiently degraded in the process of autophagy, 60,61 were notably increased dosedependently in the BA treatment group compared with the control group, and SQSTM1 knockdown in HCT116 cells hadn't an obvious influence on BA induced the conversion of LC3-I to LC3-II (Supplementary Figure S3), suggesting that BA-induced autophagy was involved in accumulation of SQSTM1, whereas SQSTM1 did not affect BA-induced autophagy.
Blockage of autophagy enhances BA-induced proliferation inhibition and apoptosis in CRC cells. To determine the role of autophagy on BA-induced antitumor effect in CRC cells, we inhibited autophagy by autophagy key gene silencing method or administration of autophagy inhibitors. Results showed gene silencing of autophagy key gene ATG5 or administration of autophagy inhibitors chloroquine notably enhanced BA-induced proliferation inhibition and apoptosis in CRC cells (Figure 4 and Supplementary Figures S4 and S5). Consistently, the cleavage of pro-apoptosis protein PARP was also enhanced in ATG5 silencing or CQ treatment in contrast to the control (Supplementary Figure S5). Besides that, another autophagy key gene BECLIN1 silencing and cotreatment with another autophagy inhibitor 3-MA, an inhibitor    Figure S5). Taken together, these results demonstrate that autophagy has a protective role in BA-treated CRC cells.
The AKT-MTOR signaling pathway was involved in BAinduced autophagy. The AKT-MTOR signaling is reviewed as a key negative regulator of autophagy. 63 Therefore, we verified whether BA-induced autophagy is also the case in CRC cells. As shown in Figure 5a, BA treatment inhibited phosphorylation of both AKT (S473) and MTOR (S2448) in HCT116, SW480 and HT29 cells. In addition, overexpression of continuous activation Myr-AKT-delta vector decreased BAinduced conversion of LC3 contrast with control in three kinds of CRC cells (Figure 5b), suggesting that BA could induce autophagy by inhibiting AKT-MTOR signaling in CRC cells.
BA first activated and then rapidly degraded p53 by the ubiquitin-proteasome pathway in mutp53 and wtp53 harboring CRC cells. To analyze why and how BA affects p53 expression, three kinds of CRC cells harboring either mutant p53 (SW480 and HT29) or wild-type p53 (HCT116) were analyzed by immunoblotting. As shown in Figure 1d, BA strongly reduced the levels of mutp53 and wtp53 protein, but increased their mRNA levels and the mRNA expression of wild-type p53-targeted genes (such as NOXA, PUMA, P21 and BAX; Figure 6a), suggesting that BA can enhance p53 degradation. To investigate the mechanisms of BA-induced p53 degradation, we next performed cycloheximide chase experiment. As shown in Figure 6b, BA markedly decreased the half-life of both wtp53 and mutp53 proteins. However, proteasome inhibition by MG132 rescued BA-induced degradation of wtp53 and mutp53 ( Figure 6c). In addition, autophagy inhibition did not affect BA-induced p53 protein degradation and MDM2 protein was also decreased by BA ( Supplementary Figures S5A and B), indicating that BAinduced p53 protein degradation is not dependent on autophagy regulation and its negative regulator, the E3 ubiquitin ligase MDM2. Taken together, our results indicated that BA degraded both wild-type p53 and mutant p53 protein by ubiquitin-proteasome pathway independent of MDM2. Owing to that BA can induce transcription of p53 and activation of p53 target gene, p53 may be activated and involved in regulating CRC apoptosis in the early stage of BA treatment. To this end, we examined a short kinetic of p53 quantity after BA treatment for 0, 4, 8, 12, 24 and 48 h. Results showed that BA induced p53 expression and activation till 12 h in HCT116 and HT29 cells and till 4 h in SW480 cells, and then rapidly degraded p53 in three CRC cells (Figure 6d; Etoposide was used as a positive control to activate p53 in CRC cells (Supplementary Figure S6)), suggesting that short exposure to BA firstly activates p53 and then rapidly degrades p53 by the ubiquitin-proteasome pathway.     (Figures 8a-c, left) and inhibited BA-induced accumulation of GFP-LC3 puncta in CRC cells (Figures 8d-f, left). p53 overexpression increased BA-induced LC3-II expression (Figures 8a-c, middle) and GFP-LC3 puncta in HCT116 p53 − / − , SW480 and HT29 cells (Figures 8d-f, right). In addition, BA-induced LC3-II protein expression in HCT116 p53+/+ was more than that in BA-treated HCT116 p53 − / − cells (Supplementary Figure S9A) and endogenic LC3 puncta in p53-knockdown HCT116 and HT29 cells also decreased compared with the control group (Supplementary Figure S9B). To determine the role of p53 in BA-induced autophagy flux in CRC cells, a tandem monomeric RFP-GFP-tagged LC3 was used to measure the rate of delivery of autophagosomes to lysosomes. The results showed that BA increased the numbers of both autophagosomes and autolysosomes in CRC cells, which was heightened by p53 overexpression or was restored by silencing p53 (Supplementary Figure S10-S12). These results were confirmed by the evidence that p53 overexpression resulted in further increased BA-induced LC3-II conversion, while p53 silencing inhibited BA-induced LC3-II conversion in the presence or absence of CQ (Supplementary Figure S13). These results demonstrated that p53 could enhance BA-induced autophagy and autophagy flux.
Notably, low dose of ectopic p53 overexpression enhanced BA-induced protective autophagy, whereas high dose of p53 overexpression would reduce BA-induced LC3 conversion (Supplementary Figure S14). In addition, it is known that several miRNAs (e.g., miR34a, miR-218 and miR-502) were involved in p53-mediated cell cycle regulation, autophagy and apoptosis in CRC cells. [64][65][66] Interestingly, we found that BA treatment can induce miR-218 expression in three CRC cells, as well as significantly enhance miR-502 expression in HCT116 and HT29 cells and slightly accumulate miR-502 in SW480 cells (Supplementary Figure S15). These lines of evidence suggested that p53-associated non-coding miRNAs were involved in regulating BA-induced apoptosis and autophagy.
BA improves chemotherapeutic response in mutp53 CRC cells by degrading mutp53. Many studies proved that high expression levels of mutp53 proteins in cancer cells have acquired GOF that actively contribute to cancer development progression, and mutp53 depletion in human cancer cells caused chemosensitization toward an array of conventional genotoxic drugs. 44,45,67 Both BA-induced mutp53 degradation and its associated inhibition of protective autophagy can improve sensitivity of mutp53 cancer cells in response to drug treatment. Our results also found that mutp53 cells (SW480 and HT29) were much more sensitive to BA than wild-type p53 cells (HCT116; Figures 1a and b). Therefore, we asked whether pharmacological degradation of mutp53 by BA could also mediate chemosensitization in response to conventional  (Figures 9a and b). These results suggested that BA could improve other chemotherapeutic drugs partly dependent on degraded mutated p53 in CRC cells.

Discussion
BA is a bioactive pentacyclic lupine-type triterpenoid natural compound that exhibits anticancer properties. 4,6,8,9 Here, we reported that BA exerted its anticancer effect by triggering cell proliferation inhibition and apoptosis in CRC cells. In addition, BA induced a protective autophagy by inhibiting the AKT-MTOR signaling pathway, and p53 can further augment this autophagy in CRC cells. To avoid the overaccumulation of protective autophagy, BA would degrade both wtp53 and mutp53, which makes preferential cytotoxicity of BA to mutp53 cells. Autophagy is a tightly regulated catabolic process of cellular self-digestion by which cellular components are targeted to lysosomes for their degradation. 25 In response to cancer therapy, autophagy has four faces of function, which are characterized as cytoprotective, cytotoxic, cytostatic and nonprotective. 30 It was demonstrated that BA blocked autophagic flux in KM3 cells and the inhibition of autophagic flux contributed to BA-mediated apoptosis of KM3 cells. 7 In addition, autophagy was also activated as a response to the mitochondria damage inflicted by BA, which served as a rescue pathway in HeLa, MCF7, A549 and SW480 cells. 10 However, B10, a new glycosylated derivative of BA, induced autophagy and abrogated the autophagic flux. By concomitant induction of autophagy and inhibition of the autophagic flux, B10 turns autophagy into a cell death mechanism. 36 Moreover, another research showed that BA decreased phosphorylation of AKT and degraded EGFR in company with induction of autophagy in bladder cancer, which also resulted in autophagic cell death. 68 In our study, we found BA-induced p53 has been linked with regulation of autophagy. 47,51,69 However, the exact nature of this link remains seemingly controversial. It has been reported that nuclear p53 facilitates autophagy by trans-activating its target genes, whereas cytoplasmic p53 mainly inhibits autophagy by transcriptionindependent mechanisms. 52 However, these previous observations exclusively reached by p53 or mutp53 overexpression assays are not confirmed by our investigation. Indeed, we revealed that p53 overexpression enhanced BA-induced autophagy, whereas p53 depletion had the contrasting effect, which indicated p53 augmented BA-induced protective autophagy. Besides that, it is also worth mentioning another finding in our study that low overexpression of p53 increased BA-induced autophagy, whereas high overexpression of p53 decreased BA-induced autophagy (Supplementary Figure S14). It may be explained that BA cannot effectively regulate the function of p53 when exogenetic p53 overexpression exceeds a certain amount and p53 may inhibit autophagy by other signaling processes, which is not directly dependent on BA treatment. Autophagy can be regulated by p53-associated non-coding RNAs (for example, miR34a, miR-218 and miR-502). 65,[70][71][72] In this study, we found that BA treatment induced miR-218 expression in three CRC cells, whereas it significantly enhanced miR-502 expression in HCT116 and HT29 cells and slightly augmented it in SW480 cells (Supplementary Figure S15). miR-218 has been observed to induce CRC cell apoptosis by inducing p53 expression 64 and AKT-mTOR inhibition, which is a major pathway to regulate autophagy. 73 However, miR-502, which could inhibit autophagy in CRC cells, had potential p53binding sites in their putative promoter regions and was regulated by p53 via a negative feedback regulatory mechanism. 65,74 Therefore, it is possible that BA upregulates miR-218 to induce p53 within a short-time treatment and p53 degradation may increase miR-502, which will inhibit protective autophagy overaccumulation.
As we all know, wtp53 as a tumor suppressor protein has been implicated in apoptosis. 69 It was reported that BA triggered apoptosis without activation of p53 in neuroectodermal and breast cancers. 53,75 Thus, it undoubtedly prompted us  Figure S9A), which maybe because that BA activates p53 till 12 h to induce apoptosis in HCT116 p53+/+ cells and then rapidly degraded p53 to inhibit protective autophagy, but the early stage of BA-induced p53 expression has a stronger effect on apoptosis than BA-induced and p53enhanced protective autophagy. Moreover, mutp53 transfection enhanced BA-induced autophagy and inhibited the sensitivity of HCT116 p53 − / − to BA (Figures 7c and d), whereas mutp53 silencing in mutp53-harbored SW480 and HT29 cells inhibited BA-induced protective autophagy and augmented BA-induced apoptosis (Figure 7f and Supplementary Figure S7B), suggesting that this mutp53promoted and BA-induced autophagy have a role in mutp53induced BA resistance in CRC cells. In addition, BA upregulated p53 target gene in the presence or absence of p53 in mutp53 and wtp53 CRC cells (Figure 6a and Supplementary Figure S8), suggesting that alternative pathways apart from p53 are involved in BA-induced upregulation of p53 target gene. There is growing evidence that mutp53 is a clinically relevant target for intervention. 76 The hyperstability of mutp53 (including p53R273H mutants in HT29 and p53R273H/P309S mutants in SW480) is the basis for its GOF and dominant negativity that promotes malignancy and chemoresis-tance. 39,40,45,67 mutp53 is more resistant to proteasomedependent degradation compared with wtp53, but the exact identity of the pathway is still on debate. 38,40 Thus, preventing mutp53 accumulation provides an important chemopreventive and chemotherapeutic strategy. Our study showed that BA preferentially degraded mutp53 through the ubiquitin-proteasome pathway (Figure 6c), indicating that BA treatment may overcome some forms of drug resistance in mutp53 cancers. It has been reported that cytotoxic drugs are given in combinations to enhance their antitumor effectiveness during polychemotherapy. 77 To this end, we further investigated the application of BA combination in clinical therapy and found that low concentrations of BA combination with chemotherapy drugs such as 5-FU and DOX further caused cell killing than chemotherapy drug treatment alone in mutp53 cancer cells by partly inducing mutp53 degradation (Figures 9a and b), indicating that BA may have an increased therapeutic effect on mutp53-harboring cancers in clinical application.
Overall, this study highlights that BA induced a protective autophagy by inhibiting AKT-MTOR signaling. In addition, BA firstly activated and then rapidly degraded p53 to attenuate this protective autophagy, which makes BA act as one attractive drug in combination with other chemotherapy agents to treat mutp53 CRC cells ( Figure 10). Our findings revealed that BA induced CRC cell death and BA reduced the incidence of mutp53 cancer chemotherapy resistance by degrading mutp53 may have important implications if used for cancer therapy.

Materials and Methods
Cells and cell culture. Human colorectal cancer HCT116 (wtp53) cells, SW480 (p53R273H/P309S) cells and HT29 (p53R273H) cells were purchased from Shanghai Cell Bank in China. Cells were maintained in DMEM (HyClone, Logan, UT, USA) supplemented with 10% heat-inactivated fetal bovine serum (GIBCO, Carlsbad, CA, USA), penicillin and streptomycin (Hyclone). All cells were maintained in a humidified 5% CO 2 atmosphere at 37°C.  Figure 10 Model of the molecular mechanism unraveled in the presented study. BA induced a protective autophagy by inhibiting AKT-MTOR signaling and degraded p53 to attenuate this protective autophagy overaccumulation, which makes BA acting as one attractive drug in combining with other chemotherapy agents to treat mutp53 CRC cells p53-mediated autophagy protects against BA-induced apoptotic cell death S Wang et al