Quercetin enhanced the sensitivity of colorectal cancer cells to 5- fluorouracil by induction of autophagy and Drp-1 mediated mitochondrial fission

doi:10.21203/rs.3.rs-3044266/v1

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Quercetin enhanced the sensitivity of colorectal cancer cells to 5- fluorouracil by induction of autophagy and Drp-1 mediated mitochondrial fission

https://doi.org/10.21203/rs.3.rs-3044266/v1

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Background: 5-fluorouracil (5-FU) is the main chemotherapeutic agent for colorectal cancer (CRC) treatment, while the emergence of drug resistance or insensitivity hindered the clinical benefit. Thus, it is impending to explore novel effective chemotherapeutic adjuvants to increase patients' survival rate. The anticancer activity of quercetin (Que) has been reported in various of cancer such as lung, gastric, breast and pancreatic cancer. Nevertheless, the underlying anti-cancer molecular mechanism of quercetin against colorectal cancer remains to be investigated.

Methods: MTT assay and colony formation assay were performed to explore the growth inhibition of quercetin alone or combine with 5-FU on colorectal cancer cells. GFP-LC3 and mRFP-GFP-LC3 plasmids were applied to detect autophagy and autophagy flux. Hoechst staining, DCFH-DA, JC-1, Mito-Tracker red were used to evaluate apoptosis, ROS, mitochondrial membrane potential (MMP) and mitochondria fission. The expression of proteins was assessed by western blotting.

Results: The results demonstrated that quercetin inhibited cell proliferation, induced apoptosis and autophagy in CRC cells in vitro. Autophagy blockage enhanced quercetin-induced cytotoxicity, indicating that quercetin induced protective autophagy. Quercetin induced excessive ROS accumulation and decreased mitochondrial membrane potential (MMP), which were associated with the imbalance of mitochondria dynamic. Quercetin promoted Drp-1 mediated mitochondria fission eventually caused mitochondrial dysfunction and cell death. Moreover, Quercetin synergistically increased the sensitivity of colorectal cancer cells to 5-fluorouracil by induction of autophagy and Drp-1 mediated mitochondria fission. Conclusion: Our results revealed that Quercetin induced apoptosis through Drp-l mediated mitochondria fission and promoted autophagy. Furthermore, Quercetin synergistically enhanced the sensitivity of colorectal cancer cells SW480 and HCT116 to 5-FU.

Autophagy

Quercetin

Chemosensitivity

Colorectal cancer

Mitochondrial dysfunction

Colorectal cancer (CRC) is the third major diagnosed cancer and the second leading cause of cancer-related death worldwide. With over 1.9 million new colorectal cancer cases and 935,000 deaths in 2020, accounting for nearly one in 10 cancer cases and deaths[1, 2]. Currently, surgery is the preferred curative treatment, especially for early-stage tumors[3]. For advanced CRC, adjuvant chemotherapy is the main conservative treatment strategy, in spite of drug resistance[4]. Over the past decades, 5-fluorouracil (5-FU) has remained the main clinical chemotherapeutic agents for CRC treatment, while the non-specific cytotoxicity and acquired drug resistance often limited the effectiveness of chemotherapy[5]. The conception of combination therapy develops a new regimen for the drug combination of CRC[6]. Although the treatment of 5-FU in combination with other chemotherapeutic drugs such as bevacizumab, oxaliplatin or irinotecan has improved the clinical efficacy, the severe adverse effects and toxicity issue remains ineluctable[7, 8]. Hence, it is of great practical significance to develop the less toxic agents to replace chemotherapeutic drugs or as auxiliary drugs in the treatment of colorectal cancer.

Since natural products and their derivatives have strong anti-cancer effects and the potential to reverse drug resistance with minimum adverse effects, they are ideal candidate for the combination therapy to enhance the sensitivity of CRC to 5-FU. Quercetin, one of the most common natural flavonoids compounds, is found ubiquitously presented in various of fruits, vegetables and some leaves of Chinese herbal plants, and exerts multiple biological functions such as anti-inflammatory, anti-oxidant and anticancer[9, 10]. Over the last few decades, increasing studies have reported that quercetin has anticancer effects in many cancers including liver, lung, gastric, prostate, breast and ovarian cancers[9, 11–14]. It has been widely reported that quercetin exerts anti-cancer effects by inducing cell cycle arrest, apoptosis, inhibiting angiogenesis and metastasis, and mediating autophagy[10]. Moreover, quercetin has been reported to increase the chemosensitivity or reverse drug resistance of malignant tumors. Quercetin enhanced the efficacy of Doxorubicin via reducing P-glycoprotein expression[15]. Combination quercetin with irinotecan had synergistic effects on reducing the peritoneal cavity tumor numbers in Ehrlich ascites tumor-bearing mice, and decreasing the population of tumor infiltrating Tie 2-expression monocytes (TEM)s in gastric tumor specimens[16]. Nevertheless, the specific mechanisms underlying quercetin inhibiting colorectal cancer progression has not been fully understood, and the effects of combination with chemotherapeutic agents 5-FU on the chemosensitivity also need to be investigated.

To study the molecular mechanisms of the anti-cancer effects of quercetin on colorectal cancer, we treated SW480 and HCT116 colorectal cancer cell lines with quercetin alone and in combination with chemotherapeutic agent 5-FU in vitro. The results revealed that quercetin induced protective autophagy and excessive ROS accumulation in colorectal cancer cells, promoted mitochondria fission via up-regulation of Drp-1, and eventually caused mitochondria dysfunction and apoptosis. Additionally, quercetin combined with 5-FU effectively enhanced autophagy and mitochondrial dysfunction-induced apoptosis, indicated that quercetin sensitized CRC cells to 5-FU.

2.1 Cell culture, treatments and transfection

Human colorectal cancer cell line HCT116 and SW480 cells were kindly gifted from Prof. Yunlong Lei (Molecular Medicine and Cancer Research Center, Chongqing Medical University). Cells were cultured in DMEM medium (Hyclone) containing 10% fetal bovine serum (Hyclone), and 1% penicillin/streptomycin at 37°C in a humidified atmosphere with 5% CO₂. The cells were treated with quercetin alone alone or in combination with 5-FU in medium. Cells were respectively pretreated with antioxidant 5 mM N-acetylcysteine (NAC) (Beyotime, Shanghai, China), chloroquine: CQ, (Sigma-Aldrich, USA) or Mdivi-1 (Beyotime, Shanghai, China) for 30 min before administration of drugs. Thereafter, a series of analyses was performed according to the manufacturer’s instructions.

Cells were grown on coverslips in 12-well plates and incubated for 24 h to allowed to attach, and then infected with GFP-LC3 or mRFP-GFP-LC3 plasmids using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instruction. Cells were washed with PBS for twice, then disposed with Que and CQ for 24 h, and fixed with 4% of paraformaldehyde for 30 min. The fluorescent puncta in cells were observed by fluorescence microscope.

2.2 Reagents and antibodies

Reagents used were as follows: Quercetin: Que (Sigma, Q4951), 5-fluorouracil (5-FU) (solarbio, F8300), MTT (solarbio, M8180), DMSO (solarbio, F8300), Chloroquine: CQ (Sigma, C6628), Hoechst 33342 (solarbio, C0021), N-acetyl-L-cysteine (NAC) (Beyotime, S0077), Mdivi-1 (Beyotime, SC8028), Acridine orange: AO (Macklin, A6009). Quercetin and 5-FU were dissolved in DMSO, while Mdivi-1, CQ, NAC and acridine orange were dissolved in phosphate buffer saline (PBS).

Antibodies were obtained from the following sources: β-actin, Parp/Cleaved-Parp, Drp-1 were purchased from proteintech. LC3 (WL01506), P62 (WL02385), BAX(WL01637), BAK (WL0129a) and BCL-2 (WL01556) were purchased from Wanleibio. LC3 and mRFP-GFP-LC3 plasmids were kindly gifted from Prof. Yunlong Lei (Molecular Medicine and Cancer Research Center, Chongqing Medical University).

2.3 Cell viability (MTT) assay

Cell viability was measured by MTT assay as the standard protocol[17]. Briefly, colorectal cancer cells SW480 and HCT116 were seeded in 96-well plates at a density of 5×10³ in each well, and incubated for 24 h at the humidified atmosphere of 37°C, 5%CO₂. Thereafter, cells were treated with various concentrations of quercetin or 5-FU alone or in combination and incubated for 24, 48 and 72 h, and the experiment was performed in triplicate. Then 10 µL MTT (5 mg/ml) was added in each well and incubated at 37°C for 4 h. Then DMSO solution was added to dissolve the formed formazan crystals, and the optical density of each well was recorded by a microplate reader at 550 nm. The percentage of cell viability was determined by OD of treated cells/OD of untreated control cells. IC₅₀ was calculated by the proliferation inhibition curves produced by Graphpad Prism 8 computer software.

2.4 Colony formation assay

For colony formation assays, SW480 and HCT116 cells were resuspended in DMEM supplemented with 10% FBS, seeded 1500–2000 cells in 6-well plates for 24 h, and then cultured in various of quercetin and 5-FU alone or in combination for two weeks. Cells were fixed with 4% paraformaldehyde for 30 min, stained with 0.5% crystal violet solution for 15 min, washed six times, and then the colonies (> 50 cells) were estimated under a microscope.

2.5 Hoechst 33342 staining assay

Cells were plated on coverslips in 12-well plates, incubated for 24 h, and then treated with quercetin and 5-FU alone or in combination at indicated concentration for 48 h. After depletion of medium, cells were washed with PBS once and fixed by 4% paraformaldehyde for 20 min. Subsequently, Hoechst 33342 dye was added into each well for 10 min, followed by washing with PBS for 10 min twice, the blue stained nuclei were observed by fluorescence microscopy immediately.

2.6 Mitochondrial morphology

MitoTracker Green staining was used to check the morphology of mitochondria. Cells were grown on coverslips in 12-well plates and incubated for 24 h. The cells were treated with or without quercetin for 24 h. Then MitoTracker Green stock solution (1 mM) was diluted to the final working concentration (200 nM) using DMEM medium. Cells were labeled with 200 nM MitoTracker Green for 20 min at 37°C, and then washed three times. Moreover, the mitochondrial morphology was also detected when cells were exposed to quercetin with or without Mdivi-1 pretreatment. The mitochondrial morphology was captured by a fluorescence microscopy.

2.7 Reactive oxygen species (ROS) measurement

The production of intracellular ROS was measured using DCFH-DA according to the manufacturer’s instructions (Beyotime, Shanghai, China). Briefly, cells were plated on coverslips in 12-well plates incubated overnight. Then, the cells were exposed to different concentrations of quercetin with or without NAC pretreatment. Cells were then stained with 10 µM DCFH-DA and incubated for 30 min at 37°C in the dark, then washed three times with cold PBS. Eventually, the fluorescence intensity of DCFH-DA signal was captured immediately using a fluorescence microscopy.

2.8 Detection of acidic vesicular organelles (AVOs)

Acridine orange (AO) dye was used to detect the formation of acidic vesicular organelles (AVO). Cells were plated on coverslips in 12-well plates, incubated for 24 h, and then treated with quercetin or 5-FU alone or in combination at indicated concentration for 48 h. Subsequently, cells were stained with 1 mg/ml AO dye for 10 min followed by thorough washing with PBS five times. Finally, cells were observed by fluorescence microscopy to detect the AVOs which showed bright red fluoresecence.

2.9 Mitochondrial membrane potential (ΔΨm) assay

A mitochondrial-specific cationic dye (JC-1) was used to assess the influence of quercetin and 5-FU alone or in combination on mitochondrial membrane potential (ΔΨm). Cells were seeded in 6-well plates, cultured overnight, and then treated with various concentrations of quercetin for 24 h. Subsequently, cells were stained with the JC-1 dye according to the manufacturer’s instruction. Cells were incubated at 37°C in the dark for 20 min, and then washed with precooled 1× JC-1 buffer three times. The cells stained by JC-1 probe were observed using fluorescence microscopy.

2.10 Western blotting

Expressions of proteins involved in regulating autophagy, apoptosis and mitochondrial dynamics were determined by western blotting as previously described[18]. In brief, cells were washed twice with PBS and lysed in lysis buffer containing protease inhibitors. The supernatants were collected as protein samples, 30 µg protein samples were separated by 8%-12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), blocked with skim milk, then transferred to polyvinylidene difluoride (PVDF) membrane (0.22µm; Roche, Branchburg, NJ, USA) and incubated with corresponding primary antibodies (1:1500 dilution) overnight at 4°C. Subsequently, the PVDF membranes were washed thrice by TBST (0.1% Tween-20 in Tris-buffered saline), and incubated with the horseradish peroxidase-conjugated secondary antibodies (1:5000 dilution) for 2 hours at room temperature. Finally, the positive bands were visualized by enhanced chemiluminescence detection reagents (Santa Cruz). Each experiment was repeated separately for three times and the bands were analyzed by Image J software.

2.11 Statistical analysis

All experiments were independently repeated at least three times. Data were expressed as means ± SD. Statistical analysis was performed with GraphPad Prism 6.0 software. Statistic 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.

3.1 Quercetin enhanced the sensitivity of CRC cells to 5-FU

The effect of quercetin on CRC cell viability was examined by MTT assay. As shown in Fig. 1A, B, quercetin (Que) treatment significantly reduced the cell viability of colorectal cancer cells (SW480 and HCT116) in a time- and dose-dependent manner. Consistently, the proliferation of both cell lines was significantly inhibited under Que treatment as examined by colony formation assay (Figure. S1A, B). We also found the number of CRC cells decreased with Q

ue treatment and cells exhibited visible punctate vacuole formation and morphological changes in dose-dependent manner (Figure. S1C). The effect of quercetin on the apoptosis of CRC cells was evaluated by Hoechst staining. As shown in Fig. 1C, quercetin treatment induced condensed bright blue apoptotic nuclei in SW480 and HCT116 cells in a dose-dependent manner. Cells treated with quercetin were further analyzed by western blotting, and results showed that quercetin elevated the expression level of cleaved PARP (Fig. 1D-F.), a specific marker of apoptosis. Consistently, quercetin up-regulated the expression of pro-apoptotic Bax and Bak, and down-regulated anti-apoptotic Bcl-2 expression in both SW480 and HCT116 cells (Fig. 1G-L.). Collectively, these results indicated that quercetin could induce CRC cell apoptosis in vitro.

To determine whether quercetin synergized with 5-FU to induce CRC cell apoptosis. First, we checked the sensitivity of SW480 and HCT116 cells to 5-FU. As shown in Figure S1D, E, 5-FU reduced the CRC cell viability in a time- and dose-dependent manner by MTT assay. The morphology and cell number of both SW480 and HCT116 cells were changed after treatment of 5-FU for 48 h (Figure S1F). Subsequently, CRC cells were treated with quercetin alone or in combination with 5-FU. The cell viability of the combinational treatment was lower than that of the single treatment (Fig. 1M, N). Cells treated with quercetin in combination with 5-FU showed fewer colonies when compared with treated with quercetin or 5-FU alone in both SW480 and HCT116 cell lines (Figure S1G-I). Cells treated with quercetin combined with 5-FU showed more condensed bright blue apoptotic nuclei than the ones treated with quercetin or 5-FU alone (Fig. 1O). Consistently, HCT116 cells treated with quercetin and 5-FU showed elevated pro-apoptotic Bak expression and decreased anti-apoptotic Bcl-2 expression compared with the ones treated with quercetin or 5-FU treatment alone (Fig. 1P-R). Summarily, these results demonstrated that quercetin synergistically enhanced the sensitivity of colorectal cancer SW480 and HCT116 cells to tranditional chemothereapy drug 5-FU.

3.2 Quercetin induced protective autophagy and promoted autophagy flux in CRC cells

Previous study reported that quercetin induced gastric cancer cell autophagy[11]. We investigated whether quercetin could induce autophagy in CRC cells. As shown in Fig. 2A, B, quercetin treatment exhibited enhanced red fluorescence in a dose-dependent manner in SW480 and HCT116 cells by AO staining, which indicated the formation of acidic vesicular organelles (AVOs), a main feature of autophagy. An autophagy marked gene GFP-LC3 plasmid was transiently transfected to further examine whether quercetin could induce autophagy. The results showed that cells treated with quercetin exhibited more GFP-LC3-positive punctate dots compared to control cells (Fig. 2C, D). Consistently, Western blotting results revealed that the turnover of LC3-I to lipidated LC3-II was significantly increased in a dose-dependent manner (Fig. 2E), which suggested that quercetin induced autophagy in CRC cells.

Interestingly, the expression level of P62, a well-known autophagic degradation substrate, in Que-treated cells decreased along with the increased LC3-II levels (Fig. 2E), indicating that quercetin induced complete autophagic flux. Meanwhile, pre-treatment with autolysosome inhibitor chloroquine (CQ) resulted in an accumulation of endogenous LC3 puncta (Fig. 2C, D). To further investigated whether quercetin promoted the progress of autophagy flux, a kind of tandem monomeric RFP-GFP-tagged LC3 plasmids was transiently transfected into cells, which contained acid-sensitive GFPs and acid-insensitive RFPs. Results showed that quercetin treatment induced remarkable formation of red fluorescent dots, indicating that quercetin induced the fusion of neutral autophagosomes and acid lysosomes (Fig. 2F). CQ pre-treatment inhibited the fusion of green fluorescence in autophagosomes and lysosomes, and these cells showed fewer red fluorescent dots (Fig. 2F). Collectively, these findings demonstrated that quercetin promoted autophagy flux in colorectal cancer cells.

Autophagy activation can promote cancer cells survival (protective autophagy), or contribute to cancer cell death (cytotoxic autophagy). To further determine the relationship between autophagy and apoptosis, we adopted MTT method to investigate cell viability after autophagy inhibition by CQ pre-treatment. The results showed that autophagy inhibition reduced cell viability, indicating that quercetin induced protective autophagy in CRC cells (Fig. 2G, H).

We further examined whether quercetin combined with 5-FU could enhance autophagy progression in CRC cells. The results showed that cells treated with quercetin comibined with 5-FU displayed more bright red fluorescent dots by acridine orange AO staining (FigureS2A, B). Next, cells transiently transfected with GFP-LC3 plasmids were treated with quercetin/5-FU/CQ alone or in combination. Results showed that 5-FU in combination with quercetin augmented more LC3 dots compared with 5-FU treatment alone, and CQ pre-treatment further resulted in the accumulation of LC3 dots (Figure S2C-F). Subsequently, RFP-GFP-LC3 was transfected into SW480 cells to further check the effect of combinatorial treatment on the autophagy flux. The results demonstrated that the combinatorial treatment increased the red fluorescent autophagosomes when compared with invidual drug treatment (Figure S2G, H), indicating that the quercetin treatment enhanced the 5-FU-induced autophagy. We also found that combinatorial treatment of quercetin and 5-FU induced protective autophagy (Figure S2I, J). In summary, quercetin induced protective autophagy and promoted autophagy flux in CRC cells.

3.3 Quercetin modulated ROS generation and reduced mitochondrial membrane potential in CRC cells

Previous study has demonstrated that quercetin modulated ROS production, eventually leading to apoptosis in prostate cancer cells[19]. In the present study, we investigated whether ROS was also involved in Que-induced apoptosis. A kind of ROS-specific fluorescent probes DCFH-DA were used to detect intracellular ROS levels in quercetin-treated CRC cells. As shown in Fig. 3A, quercetin significantly elevated intracellular ROS in a dose-dependent manner in both SW480 and HCT116 cells (Rosup as a ROS positive control), which could be scavenged by a kind of ROS scavenger, NAC (N-acetylcysteine) (Fig. 3B).

A strong correlation between ROS and mitochondrial membrane poteintial (ΔΨm) exists[20], so we further explored the effect of quercetin on ΔΨm. The lipophilic cationic probe JC-1 was used to assess the ΔΨm in CRC cells treated with quercetin, which was specific to mitochondria that could be incorporated into the mitochondrial membrane. When the membrane potential is low, JC-1 exist in the former of a monomer and emits green fluorescent light. At higher membrane potential, JC-1 aggregates increase and emit red fluorescent light. JC-1 probes can reversibly emit fluorescent light from red to green as the the ΔΨm varies. The results demonstrated a dose-dependent decreased in red fluorescence and increased in green fluorescence (CCCP-1 as a positive control), indicating that quercetin could reduce mitochondrial membrane potential (Fig. 3C). Collectively, these results showed that quercetin induced ROS accumulation and reduced mitochondrial membrane potential in CRC cells.

3.4 Drp-1 involved in quercetin-induced mitochondrial fission

The reduction of mitochondrial membrance potential is a hallmark of mitochondrial dysfunction[21]. Previous studies have shown that mitochondrial dynamics are associated with mitochondrial dysfunction [22]. We first analyzed the effects of quercetin on mitochondrial fission and fusion using a mitochondrial-specific fluorescence probe Mito-Tracker Red CMXROS. The result showed that cells treated quercetin displayed smaller and more punctate fragmented mitochondria in a dose-dependent manner both in SW480 and HCT116 cells (Fig. 4A), indicating that quercetin treatment induced aberrant mitochondrial fission and imbanlance of mitochondrial dynamics. Then we detected the key regulatory factor in mitochondrial fission, Drp-1. The result showed that quercetin treatment elevated the expression levels of Drp-1 (Fig. 4B). To further determine that the aberrant mitochondrial fission was caused by the increased Drp-1, a Drp-1 inhibitor mdivi-1 was used to specifically inhibit the expression of Drp-1. The result showed that mdivi-1 pre-treatment reversed the mitochondrial fragmentation induced by quercetin (Fig. 4C), indicating that quercetin-induced mitochondrial fission was mediated by Drp1.

To further examine the effect of combination of quercetin and 5-FU on mitochondrial function, the probe JC-1 was used to detect mitochondrial membrane potential. Cells were treated with quercetin and 5-FU in combination showed more green fluorescent dots and fewer red dots compared to that treated with quercetin or 5-FU alone (Figure S3A). Furthermore, the results showed that quercetin in combination with 5-FU increased the fragmented mitochondria and the expression of Drp-1 when compared with single treatment analyzed by Mito-Tracker red and western blotting (Figure S3B, C). Collectively, these results suggested that quercetin and 5-FU synergistically promoted aberrant mitochondrial fission via upregulation of Drp-1, contributing to mitochondrial dysfunction and eventually causing cell death. The molecular mechanisms by which quercetin enhanced the sensitivity of colorectal cancer cells to 5-FU were shown in Fig. 4D.

Drug resistance generation will lead to failure of cancer chemotherapy. Studies have shown that some Chinese herbal ingredients can increase the sensitivity of cancer cells to chemotherapy drugs[23, 24]. Quercetin, widely present in foods such as fruits, vegetables and tea, has been shown to play multiple roles in cardiovascular, neurodegenerative and cancer[9, 25, 26]. Studies connecting quercetin and cancer have shown that this kind of polyphenol play a potential anti-cancer role in multiple cancer cells, including prostate, breast, liver, lung and pancreatic cancer[27–30]. Combination with 5-FU, quercetin has been reported to play a synergistic role in cancer treatment[31, 32], However, the effect of quercetin combination with 5-FU on CRC progression is seldom report and yet to be explore. The present study first confirmed the effect of quercetin on the sensitivity of CRC cells to chmotherapy drug 5-FU and the mitochodrial dysfunction.

Previous studies have demonstrated that quercetin can inhibit proliferation via upregulation of tyrosine phosphatase receptor-type R (PTPRR) in multiple myeloma cell lines[33], and induce apoptosis cells through inhibition of Akt-CSN6-myc signaling axis in colorectal cancer HT29 cells[34]. Consistent with these results, our present study suggested that quercetin suppressed the growth and induced apoptosis in colorectal cancer SW480 and HCT116 cell lines. The levels of proteins associated with apoptosis altered in cells treated with quercetin. Quercetin decreased the expression of anti-apoptotic protein bcl-2 and increased the level of pro-apoptotic protein BAK and BAX in both SW480 and HCT116 cells. The Bcl-2 family proteins regulate the permeability of the outer mitochondrial membrane and thus lead to activation of mitochondrial intrinsic pathway of apoptosis[35]. Meanwhile, the increased level of PARP by quercetin also implies the pro-apoptotic role of quercetin, although further detailed mechanisms of quercetin-induced apoptosis need to verify.

Autophagy and apoptosis may be used as alternative and/or combined strategies adopted by cancer cells exposed to stressors[36, 37]. Accumulating evidence has shown autophagy plays a dual role in carcinogenesis, promoting cell survival or cell death[38]. The effect of quercetin-regulated autophagy on cancer chemotherapy is controversial yet[39, 40]. In present study, treatment with quercetin contributed to the formation of AVOs, increased expression of LC3-II and accumulation of green GFP-LC3 dots, indicating that quercetin induced autophagy, which coincided with previous studies[11, 41]. Moreover, quercetin treatment induced the decreased level of p62 and the accumulated red fluorescent dots of LC3, suggesting quercetin promoted autophagy flux. Further functional analysis in the study indicated that pre-treatment with CQ, a classical autolysosome inhibitor, significantly weakened Que-induced cytotoxicity, suggesting that quercetin-induced autophagy played a protective role in colorectal cancer SW480 and HCT116 cells. Quercetin induced protective autophagy in CRC cells, which was consistent with previous finding in gastric cancer[11], glioblastoma[42] and ovarian cancer [39]. Nevertheless, quercetin could enhance TRAIL-induced cell death through induce autophagy flux in lung cancer cells[43], induce cytotoxic autophagy through SIRT1/AMPK signaling pathway in human lung cancer[41], and lead to autophagic cell death via regulation of aMKKβ/AMPK/mTOR signal pathway[44]. Whether quercetin induces a protective autophagy or cytotoxic autophagy may be due to the type of cancer.

Quercetin, a common polyphenol, is well known to combate free radicals harmful to human health. However, in fact, plant polyphenols can have both anti-oxidation and pro-oxidation properties [45]. Recent studies have showed that quercetin can elevate ROS production and induce cancer cell death via ROS-dependent ferroptosis [46, 47]. Herein, we found excessive ROS accumulation in quercetin-treated cells, which could be scavenged by NAC. Increased ROS accumulation may attack the mitochondrial membrane, resulting in varied mitochondrial membrane (ΔΨm) potential and eventually contributing to mitochondrial dysfunction[20, 48]. The decrease of ΔΨm is a hallmark of early apoptosis[49], which is consistent with our study results. Mitochondria, as highly dynamic organelles, undergo coordinated balance of fission and fusion to maintain their morphology, which is referred as mitochondrial dynamics[50]. We found that quercetin treatment drived the fission of mitochondria mediated by Drp-1. Drp-1, a GTPase family memeber, was closely associated with mitochondrial morphology and function[51–53]. Furthermore, recently studies have reported that Drp1-mediated imbalance of mitochondrial dynamics can induce cell apoptosis in breast cancer[54] and lung cancer[55]. In the study, Drp-1 inhibitor Mdivi-1 rescued the fragmented mitochondria induced by quercetin, indicating that quercetin induced mitochondrial fission via up-regulation of Drp-1. Collectively, our study suggested that quercetin induced ROS accumulation and mitochondrial dysfunction through up-regulation of Drp-1 in colorectal cancer cells.

Accumulating evidence have disclosed that quercetin exerts synergistic anticancer effect in combination with chemotherapy drugs such as Paclitaxel[56], Cisplatin[57], Doxorubicin[58] and 5-fluorouraci (5-FU) [59]. In breast cancer MDA-MB-231, quercetin inhibits the metastases via regulation of MMP2 and MMP9 in combination with 5-FU[59]. Also, quercetin acts synergistically with 5-FU to promote cell apoptosis by inhibiting NF-κB in esophageal cancer cells[32]. In our current study, we demonstrated that quercetin enhanced the sensitivity of colorectal cancer SW480 and HCT116 cells to conventional chemotherapy drug 5-FU. Quercetin increased the effect of 5-FU in regard to induction of apoptosis and promotion of autophagy. Furthermore, quercetin further decreased mitochondrial membrane (ΔΨm) potential and promoted mitochondrial fission in combination with 5-FU. Therefore, quercetin might be a good candidate adjuvant chemotherapy drug to enhance the anticancer effect of conventional drug 5-FU in colorectal cancer.

In the present study, we provided evidence that quercetin exerted anticancer activity and enhanced the sensitivity of CRC cells to chemotherapeutic drug 5-FU. The link between quercetin and cancer prevention has been further strengthened. Just as most doctors and nutritionists advocate, the consumption of quercetin-rich foods, like apples and tea, may be benefical for our health and help combat cancer cells. More importantly, in the process of chemotherapy, quercetin can be used in combination with chemotherapy to increase the sensitivity of colorectal cancer patients to 5-FU.

Author contributions

J.Z. conceived and designed the study and revised the manuscript. M.L., J.X., J.F., M.H. performed the experiments, analyzed the data, and drafted the manuscript. M.L., J.X., and J.F. contributed equally to this study. All authors have read and approved the final manuscript.

Funding

This work was supported by the grants from the National Natural Science Foundation of China (82003380), the Natural Science Foundation of Chongqing (cstc2018jcyjAX0573, cstc2020jcyj-msxmX0110) and the Scientifific and Technological Research Program of Chongqing Municipal Education Commission (KJ202000541975044).

Conflicts of Interest

The authors have nothing to disclose about conflicts of interest relevant to this manuscript.

Data availability

Data will be made available on request.

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Quercetin enhanced the sensitivity of colorectal cancer cells to 5- fluorouracil by induction of autophagy and Drp-1 mediated mitochondrial fission

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Abstract

Figures

1. Introduction

2. Materials and Methods

2.1 Cell culture, treatments and transfection

2.2 Reagents and antibodies

2.3 Cell viability (MTT) assay

2.4 Colony formation assay

2.5 Hoechst 33342 staining assay

2.6 Mitochondrial morphology

2.7 Reactive oxygen species (ROS) measurement

2.8 Detection of acidic vesicular organelles (AVOs)

2.9 Mitochondrial membrane potential (ΔΨm) assay

2.10 Western blotting

2.11 Statistical analysis

3. Results

3.1 Quercetin enhanced the sensitivity of CRC cells to 5-FU

3.2 Quercetin induced protective autophagy and promoted autophagy flux in CRC cells

3.3 Quercetin modulated ROS generation and reduced mitochondrial membrane potential in CRC cells

3.4 Drp-1 involved in quercetin-induced mitochondrial fission

4. Discussion

5. Conclusion

Declarations

References

Additional Declarations

Supplementary Files

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