Tubeimoside-I sensitizes colorectal cancer cells to chemotherapy by inducing ROS-mediated impaired autophagolysosomes accumulation
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Tubeimoside-I (TBM), a plant-derived bioactive compound, shows antitumor activity in different tumors and can enhance the efficacy of chemotherapeutic agents. However, the detail mechanism underlying remains to be elucidated.
The cytotoxic potential of TBM towards CRC cells was examined by CCK8 assay, colony formation, LDH release assay, flow cytometry method and Western blots. The ROS levels, autophagy, apoptosis, chemosensitivity to 5-FU or DOX, etc. were determined between control and TBM-treated CRC cells.
In this study, we found that TBM could inhibit proliferation and induce apoptosis in colorectal cancer (CRC) cells. Intriguingly, TBM treatment could either promote autophagy initiation by ROS-induced AMPK activation, or block autophagy flux through inhibiting lysosomal hydrolytic enzymes, which leaded to massive impaired autophagylysosomes accumulation. Administration of autophagy initiation inhibitor (3-MA or selective ablation of autophagy related proteins) relieves TBM-induced CRC suppression, while combination use of autophagy flux inhibitor chloroquine (CQ) slightly augments TBM-induced cell death, suggesting that impaired autophagylysosomes accumulation contributes to TBM-induced growth inhibition in CRC cells. Notably, as an autophagy flux inhibitor, TBM works synergistically with 5-fluorouracil (5-FU) or doxorubicin (DOX) in CRC suppression.
Together, our study provides new insights regarding the anti-tumor activity of TBM against CRC, and established potential applications of TBM for CRC combination therapies in clinic.
KeywordsTubeimoside-I Autophagy ROS AMPK Chemosensitivity
Adenosine monophosphate-activated protein kinase
Green fluorescent protein
Lysosomal-associated membrane protein
Microtubule-associated protein 1 light chain 3
Poly (ADP-ribose) polymerase 1
Red fluorescent protein
Reactive oxygen species
Small interfering RNA
Although the dissemination of colonoscopy with polypectomy has led to a decline in the incidence and mortality rates, colorectal cancer (CRC) still is the third most diagnosed cancers and the second leading causes of cancer death among both men and women worldwide, with over 1.8 million new colorectal cancer cases and 881,000 deaths in 2018 [1, 2]. If CRC patients can be diagnosed and treated at localized stage, 5-year survival rate of which is about 90%, that will decline to 71 and 14% for patients diagnosed with regional and distant-stage disease [1, 3]. Adjuvant chemotherapy is the main treatment strategy for advanced CRC, however, a subset of patients eventually become relapsed because of acquired drug resistance to the current commonly used chemotherapeutic drugs and the effectiveness of chemotherapy has often been limited [4, 5]. Hence at present, development of new drugs for colorectal cancer is an urgent need.
The stem tuber of Bolbostemma paniculatum (Maxim.) Franquet (Cucurbitaceae) is a traditional Chinese medicine named as Rhizoma Bolbostemmatis (Chinese name“Tu Bei Mu”) [6, 7]. It was listed in the Supplement to the Compendium of Materia Medica in Qing Dynasty for treating breast cancer, acute mastitis, inflammation and snake venoms [6, 7]. Tubeimoside-I (TBM), a triterpenoid saponin, is isolated from Rhizoma Bolbostemmatis and shows antitumor activity in different tumor such as promyelocytic leukemia, lung cancer, cervical cancer, nasopharyngeal carcinoma, esophageal carcinoma with low toxicity [6, 7]. TBM could trigger a mitochondrial-related apoptotic pathway and cell cycle arrest in cervical carcinoma, ovarian cancer, choriocarcinoma and glioma [8, 9, 10, 11]. TBM can also inhibit the growth and invasion of CRC cells . More interestingly, tubeimosides are effective in combination therapies, particularly at targeting drug-resistant cancerous cells . However, the underlying mechanisms of its anti-tumor activity have not been fully clarified so far, especially in CRC.
Autophagy is a highly conserved catabolic process during which de novo-formed membrane-enclosed vesicles engulf damaged or senescent organelles and transport to lysosomes for degradation and recycling in response to nutrient starvation or metabolic stress [14, 15]. Autophagy plays an important role in the regulation of cancer progression and in determining the response of tumor cells to stress induced by chemotherapy [14, 15]. Four functional forms of autophagy induced by chemotherapy have been described to date: the cytoprotective autophagy which facilitates the resistance of cancer cells to chemotherapeutic drugs, cytotoxic autophagy which promotes cell death, cytostatic autophagy which prolongs growth inhibition as well as reduced clonogenic survival, and nonprotective autophagy which don’t affect drug or radiation sensitivity . Considering the crucial roles during chemotherapy, targeting the autophagy process has recently attracted considerable attention to develop novel antitumor therapeutics via pharmacological modulation of autophagy. Tubeimoside-I can induce cytoprotective autophagy in human breast cancer cells in vitro, while promote cytotoxic autophagy in cervical cancer cells [17, 18]. However, the role of autophagy in TBM-treated CRC cells remains largely unexplored, let alone the underlying mechanisms.
In this study, we found that TBM inhibited the growth of CRC cells by stimulating apoptosis. Interestingly, TBM elicits autophagy by ROS-induced activation of AMPK and blocks autophagic flux by impairing the degradation of the autophagolysosome, which contributes to TBM-induced anti-cancer activity. Notably, as an autophagy modulator, TBM synergistically suppresses CRC cell growth with 5-FU or DOX. These findings provide the evidences for the use of TBM as a new therapeutic agent against CRC, especially in combination chemotherapy.
Material and methods
The SW480 and SW620 cell lines were purchased from the American Type Culture Collection (ATCC), HCT116 and RKO cell lines were purchased from Shanghai cell bank. All cell lines were cultured according to the guidelines and were maintained in DMEM (Gibco) supplemented with 100 U/mL penicillin (Sigma), 10 mg/L streptomycin (Sigma), and 10% serum (Hyclone) in a humidified incubator at 37 °C under 5% CO2 atmosphere.
Reagents and antibodies
The following primary antibodies were used: Lamp1 (Santa Cruz Biotechnology), LC3 (MBL International Corporation), p62 (MBL International Corporation), PARP 1(Cell Signaling Technology), PRKAA/AMPK (Cell Signaling Technology), phosphor-PRKAA/ AMPK(Cell Signaling Technology), Beclin1 (Cell Signaling Technology), ATG5 (Cell Signaling Technology), CASP9 (Cell Signaling Technology), CASP3 (Cell Signaling Technology) and Cleaved-CASP3 (Cell Signaling Technology). TBM (BP1415) was purchased from Phytopurify Biotechnology. 3-methyladenine (3-MA), chloroquine (CQ) and N-acetyl-L-cysteine (NAC) were purchased from Sigma-Aldrich. Doxorubicin (DOX, HY-15142A) and 5-fluorouracil (5-FU, HY-90006) were from MedChem Express.
All siRNAs were designed using BLOCK-iT™ RNAi Designer (Invitrogen) and synthesized by GenePharma (Shanghai, China). The sequences of the siRNAs used are listed in Additional file 1: Table S1. Cells were transfected with siRNAs using Lipofectamine RNAiMax (Invitrogen) according to the manufacturer’s instruction.
Dominant-negative AMPK (DN-PRKAA1/DN-AMPKα1) plasmid was obtained as described previously . The plasmids were transfected into the indicated cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instruction.
Cells grown on coverslips that treated with TBM for 24 h were fixed with 4% paraformaldehyde (Sigma) for 30 min and then washed three times with PBS. Fixed cells were permeabilized with 0.4% Triton 100 and 5% BSA for 1 h at 37 °C. For staining, cells were incubated with primary antibodies for 12 h at 4 °C, followed by incubation with secondary antibodies (Thermos Fisher Scientific) for 1 h at 37 °C. Finally, Nuclei were stained with DAPI for 10 min. For autophagy flux studies, cells were transfected with GFP-RFP-LC3 for 24 h and then treated with TBM for another 24 h. Images were captured using a confocal microscopy (Carl Zess Micromaging).
Cell viability and proliferation assays
As described previously , CCK8 assay was performed to determine cell viability. Briefly, cells were seeded in 96-well plates at a density of 5000 cells. After treatment, CCK8 (Dojindo, Kamimashiki-gun, Kumamoto, Japan; CK04) were added and incubated for 40 min. The absorbance value was then determined at 450 nm.
The long-term effects of TBM on CRC cell proliferation were analyzed with a colony formation assay. After treatment, 1500–2000 cells were seeded in six-well plates and the medium was changed every 3 days. After two weeks, cells were fixed with 4% paraformaldehyde (Sigma) for 30 min and stained with Crystal Violet for another 30 min, then the colonies were washed three times and taken photos.
Immunoblot and immunoprecipitation
Cells were lysed with RIPA buffer (50 mM Tris base, 1.0 mM EDTA, 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 1% sodium deoxycholate, 1 mM PMSF) supplemented with protease inhibitor cocktail (Sigma) and then protein lysates were centrifuged and boiled with loading buffer. For immunoprecipitation, Whole cell lysates were prepared in RIPA buffer (40 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% Nonidet P-40, cocktail, 5% glycerol, 10 mM NaF) and incubated with 1 μg of antibody overnight at 4 °C. Next day, Sepharose protein A/protein G beads were added for 2 h. The immune-complexes were then centrifuged and washed 3 times using RIPA buffer. All lysates were quantified by the BCA Protein Assay (Thermo Fisher Scientific) and analysed by SDS–PAGE.
Transmission electron microscopy
Transmission electron microscopy assay was used to visualize autophagic vesicles. After 24-h TBM treatment, SW480 and HCT116 cells were fixed in glutaraldehyde (Sigma) and ultrathin sections were prepared using a sorvall MT5000 microtome. Then, the sections were stained by lead citrate and /or 1% uranyl acetate and visualized by Philips EM420 electron microscopy.
As described previously , the indicated cells treated with TBM for 24 h were harvested and washed three times with PBS, then resuspended and incubated with PI-ANXA5 solution (KeyGEN Biotech). Apoptosis was analyzed with a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA USA).
Reactive oxygen species (ROS) measurement
Intracellular ROS level was detected by staining cells with 2′, 7′-dichlorofluorescein diacetate (DCFH-DA) (GENMED, GMS10016.2) according to the manufacturer’s instructions. The DCFH-DA signal was measured with a FACSCalibur flow cytometer or a Molecular Devices SPECTRAMAX M5 fluorimeter (490 nm excitation and 530 nm emission).
Data analysis and statistics
Data were expressed as means ± s.d. All experiments were performed at least three times. Statistical analysis was performed with GraphPad Prism 6.0 software. Statistical differences between groups were determined using two-tailed Student’s t-test. Significance was designated as follows: *, P < 0.05, **, P < 0.01, ***, P < 0.001.
TBM inhibits proliferation and induces apoptosis in CRC cell
TBM activates autophagy in CRC cell
ROS/AMPK axis contributes to TBM-induced autophagy initiation
TBM impairs autophagy flux in CRC cell by inhibiting proteolytic activity of lysosome
Autophagy flux signified autophagosomes fuse with lysosomes for further degradation, thus dysfunction of lysosomes may play an important role in inhibition of TBM-induced autophagy flux. We firstly detected the expression of lysosome- associated membrane protein (LAMP) 1, a marker for mature lysosome. As shown in Fig. 5f, TBM significantly up-regulated of LAMP1 in a dose dependent manner, indicating that TBM promote lysosomal mature. We subsequently examined whether autophagosome-lysosome fusion was affected by TBM by assessing the colocalization of endogenous LC3 with LAMP1. However, our results also showed that TBM treatment induced obvious colocalization of LC3 with LAMP1, suggesting the fusion of autophagosome with lysosome (Fig. 5g). Finally, we compared the lysosomal hydrolytic enzymes after TBM treatment by examining the level of ubiquitinated proteins. Interestingly, ubiquitinated protein conjugates was increased following TBM treatment (Fig. 5h). These results suggested that TBM impairs autophagy flux by inhibiting proteolytic activity of lysosome.
ROS-induced impaired autophagolysosomes accumulation contributes to TBM-induced cell death
TBM enhances the chemotherapeutic response of CRC cells response to 5-FU and DOX
Recently, TBM has been found to have considerable potential to inhibit various types of cancer . Multiple mechanisms of TBM have been uncovered recently, including the modulation of multiple enzyme activities and signaling pathways . It is of great interest to reveal novel activities and mechanisms of TBM in cancer treatment. In the present study, we demonstrated that TBM exhibited potent antitumor effect on CRC. TBM inhibited the growth of CRC via inhibition of cell proliferation and induction of apoptosis. Interestingly, we also found that TBM was a potent autophagy modulator in CRC cells. TBM initiates autophagy by activating ROS-AMPK signaling and impairs autophagy flux through inhibiting lysosomal proteolysis activity, leading to the rough accumulation of autophagolysosomes in CRC cells. Meanwhile, our findings indicated that accumulation of immature autophagolysosomes by TBM triggered apoptotic cell death. Our results also provided the support that low concentration of TBM synergizing with 5-FU and DOX shows preferential cytotoxicity compared with treatment alone.
Autophagy is a tightly-regulated catabolic process for clearing damaged or long-lived proteins and organelles, and plays important roles in response to various stresses including cancer treatment [14, 15, 16]. The role of autophagy in response to chemotherapy is multifaceted [14, 15, 16]. On one hand, autophagy usually play a supportive role to hamper the anti-cancer effectiveness and induce drug resistance by preserving the organelle function and eliminating cellular waste products [14, 15, 16]. On the other hand, autophagy also can be pro-death or inhibit proliferation to benefit the anticancer outcome [14, 15, 16]. In this study, we found that TBM could significantly induce autophagy initiation and promote the fusion of autophagosome with lysosome, while impairs the degradation ability of autophagolysosome, leading to the robust accumulation of autophagolysosomes and autophagic cargo proteins (p62). The inhibition of autophagic flux is harmful to cancer cells in chemotherapy and has potential for clinical benefit. Indeed, inhibition of autophagy initiation could counteract TBM-induced cytotoxicity, while impeding autophagy flux by CQ would further promote cell death slightly in CRC cells, suggesting autophagy account for the anti-cancer activity of TBM in CRC cells.
Accumulating evidence has shown that autophagy can give cancer cells resistant ability to cancer treatment including chemotherapy and radiotherapy, whereas inhibition of autophagy could enhance the efficacy of anticancer agents [14, 24, 25]. Targeting the autophagy process has been regarded as a novel therapeutic approach, and autophagy inhibitors including chloroquine (CQ) and its derivative hydroxychloroquine (HCQ) have been applied in clinic [14, 24, 25]. Thus, development of novel autophagy modulator is worthwhile to be exploited. The classical chemotherapeutic drug 5-FU and DOX have exhibited compromised treatment outcome in CRC treatment due to protective autophagy initiation [26, 27, 28]. Thus, several efforts have been made to explore new combinational therapies with autophagy inhibitor [26, 27, 28]. TBM has been reported to enhance the efficacy of many chemotherapeutic agents in cancer cells. For example, TBM has the capability to increase the sensitivity of cisplatin (CDDP)-resistant human ovarian cancer A2780/DDP cells and Hela cells toward CDDP-induced cell death [18, 29], while the combination of TBM with fuzi extracts (Radix Aconiti Lateralis Preparata) exerted synergistic effects on breast cancer MDA-MB-231 and SKBR3 cells proliferation and migration . Therefore, we determined the possibility that TBM addition might sensitive CRC cells to 5-FU or DOX treatment. The present study shows that low-dose TBM in combination 5-FU or DOX exhibit good synthetic lethal effect in CRC cells, supporting that combinational treatment of TBM with conventional anticancer therapies may warrant consideration for clinical trials by modulation of autophagy.
Reactive oxygen species (ROS) is a collective term that encompasses one or more unpaired electrons of oxygen, including superoxide (O2−), hydrogen peroxide (H2O2) and the hydroxyl radical (HO•) [31, 32]. Excessive production of ROS cause biomolecules damage and even cell demise, whereas moderate levels of ROS can act as a second-messenger to regulate pro-survival pathways [31, 32]. ROS have been widely reported to play a pivotal role in apoptosis and autophagy upon cancer treatment . Major sources of cellular ROS are generated from mitochondrial respiration, NADPH oxidases (NOXs), peroxisomal β-oxidation and endoplasmic reticulum (ER) [31, 32]. TBM has been shown to induce mitochondrial damage and ROS generation in several cancer cells including hepatoma HepG2 cells, prostate cancer DU145 cells, lung cancer A549 and PC9 cells, as well as glioma U251 cells, which was involved in TBM-induced cell death [10, 13, 34, 35, 36]. In this study, we also found that TBM can promote ROS production, which in turn induces autophagy initiation by activating AMPK.
AMPK has been well characterized to play an essential role in the induction of autophagy response to cancer treatment and could be regulated by redox modification under oxidative stress [19, 37, 38]. AMPK can trigger autophagy through regulation of early autophagic events, such as activating the proautophagic VPS34 complex by phosphorylating S91/S94 in beclin1, releasing ULK1 from mTORC1 or directly activating ULK1 by phosphorylation [19, 37, 38]. AMPK is a metabolic checkpoint and regulated by energy supply, and mitochondria are the most important organelles for energy production [19, 37, 38]. TBM induced apoptosis mainly via mitochondrial pathway which will damage the mitochondria and decrease the production of energy. These may lead to AMPK activation and subsequently autophagy initiation to protect cells . Our study reveals that TBM strongly increases Thr172 phosphorylation of AMPK in a dose-dependent manner, which is crucial for the initiation of autophagy in TBM-treated CRC cells.
In summary, we demonstrate that TBM exerts strong cytotoxicity by inducing apoptosis in this study. Moreover, we revealed that TBM could promote impaired autophagolysosomes accumulation by both enhancing autophagy initiation and disrupting the autophagy flux, which is contributed to the anticancer activity of TBM. In addition, as a specific autophagy modulator, TBM could act as one attractive drug in combining with other chemotherapy agents to treat CRC cells for overcoming drug resistance inducing by protective autophagy. Our findings provide a bold therapeutic strategy to potential use of TBM as an adjuvant for further cancer treatment, especially in CRC.
Conception and design: YLL, JHY; Acquisition of data: JHY, XYD, JZ, YFX, LM and LHL; Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): JHY and JZ; Writing, review, and/or revision of the manuscript: YLL, LHL and JHY; Study supervision: YLL. All authors read and approved the final manuscript.
This work was supported by grants from the Chinese NSFC (31400999, 81872014), and Scientific and Technological Research Program of Chongqing Municipal Education Commission (Grant No. KJQN201800417, KJQN201800429).
Ethics approval and consent to participate
This article does not contain any studies with human subjects and animals performed by any of the authors.
Consent for publication
The authors agree for publication.
Jianghong Yan, Xiaoyun Dou, Jing Zhou, Yuanfeng Xiong, Ling Mo, Longhao Li and Yunlong Lei declare that they have no competing interests.
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