Polypeptide LTX‐315 reverses the cisplatin chemoresistance of ovarian cancer cells via regulating Beclin‐1/PI3K/mTOR signaling pathway
Abstract
Objective: Polypeptide LTX‐315 induces immunogenic cell death, thus having the potential to improve the effect of anticancer treatment. However, the function of LTX‐315 in reversing chemoresistance in ovarian cancer (OC) still remains elusive.Our study aims to decipher the effect of LTX‐315 on reversing the chemoresistance of OC cells and explore its mechanism.
Methods: SKOV3, A2780, SKOV3/DDP, and A2780/DDP cells (cisplatin [DDP]‐ resistant cells] were treated with different concentrations of LTX‐315 (10 and 20 µmol/L), respectively. Cell counting kit‐8 assay, Transwell assay, and flow cyto- metry were used to assess cell viability, migration, invasion, apoptosis rate, and cell cycle of the cells. Western blot was performed to examine the expression of cleaved caspase 3, caspase 3, cleaved Poly (ADP‐ribose) polymerase (PARP), PARP, Bax, Bcl‐2, Beclin‐1, p‐Akt, Akt, p‐mammalian target of rapamycin (mTOR), and mTOR.Furthermore, OC cells were treated with autophagy inhibitor 3‐methyladenine (3‐MA), and “rescue experiments” were performed.
Results: DDP‐resistant OC cell models were established, and LTX‐315 treatment resulted in lower IC50 of DDP. In OC cells treated with LTX‐315, the viability, migration, invasion and the expression of Bcl‐2 of were repressed, but the apoptotic rate and the expression of cleaved caspase 3, cleaved PARP and Bax were increased, and the cell cycle was arrested. Moreover, LTX‐315 promoted Beclin‐1 expression level and inhibited p‐Akt and p‐mTOR expression levels, whereas 3‐MA could partially reverse the biological effects of LTX‐315 on OC cells.
Conclusion: LTX‐315 can inhibit the resistance of OC cells to DDP in vitro and plays a role by regulating Beclin‐1/phosphatidylinositol‐3‐kinase/mTOR signaling pathway.
KEYWORDS : autophagy, cisplatin chemoresistance, LTX‐315, ovarian cacner
1 | INTRODUCTION
Ovarian cancer (OC) is one of the leading cancer‐related causes among women worldwide; in 2017, the incident cases and death cases of OC are 286.1 and 176.0 thousand worldwide, respec- tively.[1] Due to its insidious early clinical symptoms, more than 70% of the patients are in the advanced stage when first diagnosed, with their 5‐year survival rate merely 15%–20%.[2] Platinum‐based chemotherapy is currently the recommended chemotherapy regimen for treating OC patients with metastasis.
Nonetheless, almost all of the patients will suffer disease pro- gression due to chemoresistance.[3] Therefore, exploring under- lying mechanisms of chemoresistance in OC and identifying novel sensitizers are of substantial value for improving the prognosis of patients with OC.
LTX‐315 is an oncolytic peptide with strong immunoregulatory properties by inducing rapid immunogenic cell death through the release of danger‐associated molecular pattern molecules (DAMPs).[4] Previous studies have demonstrated that LTX‐315 promotes the release of adenosine triphosphate, cytochrome C, and high mobility group box protein 1, thus causing plasma membrane de- struction and cell death.[5] In addition, it has been previously reported that LTX‐315 impairs the function of mitochondria, dissipates mitochondrial transmembrane potential, and induces the release of mitochondrial membrane protein into cytoplasm, thus regulating the permeability of mitochondrial membrane and killing cancer cells.
Reportedly, LTX‐315 shows the therapeutic potential in a triple‐negative breast cancer and skin cancer.[4,7] However, the function and regulatory mechanism of LTX‐315 in reversing chemoresistance in OC cells still remains elusive.Beclin‐1/phosphatidylinositol‐3‐kinase (PI3K)/mammalian target of rapamycin (mTOR) signaling pathway plays a key role in regulating cisplatin (DDP) resistance in cancer cells.[8] For in- stance, activated Akt and mTOR contributes to the resistance of gastric cancer cells to DDP via repressing autophagy.[9] Pre- clinical models provide a better understanding of tumor biology and drug development to elucidate drug resistant mechanisms of tumors.[10,11] In the present work, we established DDP‐resistant cell models and investigate the effect of LTX‐315 on DDP resistance of OC cells. Our data suggest that LTX‐315 is a pro- mising anticancer agent to repress the malignant phenotyeps of OC cells, and can reverse the DDP resistance, probably via reg- ulating Beclin‐1/PI3K/mTOR signaling pathway.
2 | MATERIALS AND METHODS
2.1 | Cell culture and establishment of DDP‐ resistant cell strains
OC cell lines SKOV3 and A2780 used in this study were purchased from Type Culture Collection of Chinese Academy of Sciences. The cell line identities were authenticated through short tandem repeat profiling (LGC Standards). SKOV3 and A2780 cells were routinely cultured at 37°C with 5% CO2 using Dulbecco’s modified Eagle medium (DMEM) (Hyclone) containing 10% fetal bovine serum (FBS) (Thermo Fisher Scientific). The cell culture was confirmed to be free of mycoplasma infection using the MycoAlert Mycoplasma Detection Kit (Lonza). To establish DDP‐resistant cell lines, SKOV3 and A2780 cells in the logarithmic growth phase were cultured in DMEM con- taining DDP (Sigma‐Aldrich), and gradually increasing DDP con- centration (0, 2, 4, 8, 16, 32, and 64 µM) was used for intermittent induction in sequence. Finally, the strains capable of completely tolerating 64 µM DDP were obtained and named SKOV3/DDP and A2780/DDP, respectively.
2.2 | Cell counting kit‐8 (CCK‐8) assay
The cell viability was detected by CCK‐8 test. SKOV3 and A2780 cells or SKOV3/DDP and A2780/DDP cells in the logarithmic growth phase were transferred into 96‐well plates at a density of 4× 103 cells/well (100 μl of cell suspension in each well), with three replicated wells in each group. When the cells adhered to the bottom of the wells, the medium was replaced with fresh medium containing different concentrations of LTX‐315 (10 or 20 µmol/L) (Sigma‐Aldrich), while the blank control group was added with a culture solution containing the same volume of dimethylsulfoxide (DMSO). After continuous culture for 24, 48, and 72 h, respectively, 10 μl of CCK‐8 reagent (Dojindo) was added to each well. After 2 h of incubation, the absorbance (optical density [OD]) value of each well was measured at 450 nm by an automatic microplate reader. The average OD value of three wells was taken to indicate the cell viability, and the drug con- centration corresponding to the 50% inhibition rate of the cell viability was taken as the IC50 value.
2.3 | Transwell experiment
The invasion experiment was conducted by Transwell chambers coated with Matrigel (8 µM poresize; Corning), and Matrigel was not used in the migration experiment. In the experimental group, OC cells were treated with different concentrations of LTX‐315 for 48 h; in the control group, equal volume of DMSO was added. After LTX‐ 315 treatment, SKOV3 and A2780 cells were dispersed with 0.25% trypsin, harvested, centrifuged, and resuspended with serum‐free medium to prepare single cell suspension. Then the cells were seeded in the upper chamber at a density of 2 × 105 cells/well, while 400 μl of medium containing 10% FBS was added in the lower chamber.After incubation at 37°C for 24 h, the cells that failed to migrate or invade were removed from the membrane. Cells remaining on the below surface of the Transwell membrane were fixed with 4% par- aformaldehyde for 10 min and stained with 0.5% crystal violet so- lution. After rinsed by tap water and dried, the cells were counted under an inverted microscope.
2.4 | Detection of cell cycle
Flow cytometry analysis method was performed to detect the apoptotic rate with AnnexinV‐fluorescein isothiocyanate (FITC)/propidium iodide (PI) double staining kit (Yeasen Biotech Co., Ltd). SKOV3, A2780, SKOV3/DDP, and A2780/DDP cells in the logarithmic growth phase
were harvested to prepare single‐cell suspension and a total of 4× 105 cells/well were transferred into six‐well plates. After 24 h of culture, the medium in experimental group was replaced with DMEM containing different concentrations of LTX‐315 (10 and 20 μmol/L), while
the blank control group was replaced with DMEM containing DMSO with the same volume. After continuous culture for 24 h, the cells were harvested, and resuspended in binding buffer (1 × 105 cells in 100 μl of binding buffer for each sample). A volume of 5 μl AnnexinV‐FITC and 10 μl PI staining solution were then added to each sample, respectively, then the cell suspension was incubated at room temperature in the dark for 15 min. Then the cell suspension was added with binding buffer until the total volume reached 500 μl, and after that, the apoptosis of the cells were detected by a FACSCanto II flow cytometer (BD Biosciences).
2.5 | Detection of cell cycle
For cell cycle analysis, the cells were collected and fixed using 70% ethanol. After washing with phosphate buffered solution (PBS) and subsequently washing with staining buffer, for each sample, 1 × 106 cells were resuspended in 0.5 ml of PI/RNase Staining Buffer (BD Biosciences), and cells were incubated for 15 min at room tempera- ture in the dark. Subsequently, a FACSCanto II flow cytometer (BD Biosciences) was used to analyze cell cycle distribution.
2.6 | Western blot
Western blot was performed to examine the expression level of cleaved caspase 3, caspase 3, cleaved Poly (ADP‐ribose) polymerase (PARP),
PARP, Bax, Bcl‐2, Beclin‐1, p‐Akt, Akt, p‐mTOR, and mTOR proteins. The cells were divided into control group (wild type cell lines), DDP‐resistant cell line group, LTX‐315 treatment group (20 µmol/L) and 3 methyladenine (3‐MA) treatment group (2 mM). Cells in the logarithmic growth phase were inoculated into six‐well plates with 2 ml suspension (about 4 × 105 cells) per well, and placed in an incubator until the cells adhered to the wall. After 48 h of cell treatment, the plate was taken out. The cells were trypsinized and collected, washed with precooled PBS, and then radioimmunoprecipitation assay lysis buffer (Beyotime Biotechnol- ogy) containing protease inhibitor was added to extract the total protein on ice for 30 min. The cells were then scraped off, placed in EP tube, centrifuged for 10 min, and after that, the supernatant was collected. Protein concentration was determined by bicinchoninic acid method and mixed with loading buffer, and the samples were boiled for denaturation and were subjected to sodium dodecyl sulphate–polyacrylamide gel electrophoresis. Subsequently, the protein on the gel was transferred to polyvinylidene fluoride membrane by wet transfer method. Following that, the membrane was blocked with blocking solution, and then the primary antibodies were added to incubate the membrane overnight at 4°C. Next day, after the membrane was washed with Tris‐buffered salin Tween 20 (TBST), the membrane was incubated with the secondary antibody for 2 h at room temperature. After that, the membrane was washed with TBST again, and ECL chemiluminescent reagent (Millipore) was added to visualize the protein bands. Antibodies used in this study were obtained from Abcam and Cell Signaling Technology, including anti‐Beclin 1 (Abcam, ab207612, 1:1000), anti‐Akt (Abcam, ab8805, 1:500), anti‐p‐Akt (Abcam, ab38449, 1:500), anti‐mTOR (CST, 2983, 1:1000), anti‐p‐mTOR (CST, 5536, 1:1000), anti‐cleaved caspase‐3 (Abcam, ab32042, 1:1000), anti‐caspase‐3 (Abcam, ab32351, 1:1000), anti‐ cleaved PARP (Abcam, ab32561, 1:1000)，anti‐PARP (Abcam, ab191217, 1:1000), anti‐Bcl‐2 (Abcam, ab185002, 1:1000), anti‐Bax (Abcam, ab32503, 1:1000) and anti‐GAPDH (Abcam, ab181602, 1:3000) (as loading control).
2.7 | Statistical analysis
All the experiments were performed in triplicate, and repeated for at least three times. All the measurement data in this study were ex- pressed as “mean ± SD.” SPSS 22.0 (IBM, SPSS) and Graphpad Prism 8.0 (GraphPad Software Inc.) were used for analyzing the data and drawing the figures, respectively. Independent sample t test was used for comparing the parameters between two samples. The dif- ference is statistically significant with p < 0.05. 3 | RESULTS 3.1 | Establishment of DDP‐resistant OC cell lines DDP‐resistant cell lines SKOV3/DDP and A2780/DDP were estab- lished by culturing SKOV3 and A2780 cells in the medium containing increasing concentrations of DDP (0, 2, 4, 8, 16, 32, and 64 µM) (Figure 1A). To confirm the drug resistance of these cells, the cell viability of the above cells was examined by the CCK‐8 method at different DDP treatment concentrations, and the IC50 value of DDP was calculated. The data indicated that the IC50 values of DDP were remarkably higher in both SKOV3/DDP and A2780/DDP cells com- pared with those in the parental cell lines (Figure 1B), indicating that DDP‐resistant SKOV3/DDP and A2780/DDP cells were successfully established. 3.2 | LTX‐315 inhibits the viability of wild type cells and DDP‐resistant cells To probe the effect of LTX‐315 on the viability of SKOV3, A2780, SKOV3/DDP, and A2780/DDP cells, the above cells were treated with different concentrations of LTX‐315 (control, 10 and 20 μmol/L) for 24, 48, and 72 h, respectively, and the cell viability was detected by CCK‐8 method. The data unveiled that the viability of SKOV3 and A2780 cells, as well as SKOV3/DDP and A2780/DDP cell, was markedly suppressed after 10 and 20 μmol/L LTX‐315 treatment compared with that in the control group, in a does‐dependent manner (Figures S1A and 2A). The IC50 value of DDP in each group was then examined, and the data showed that LTX‐315 treatment reduced the IC50 of OC cells in both wild type and DDP‐resistant cell lines (Figures S1B and Figure 2B), suggesting LTX‐315 and DDP had synergistic effect to kill OC cells. Additionally, the effects of LTX‐315 on the migration, invasion, apoptosis, and cell cycle of SKOV3 and A2780 cells were investigated. Transwell assays and flow cytometry results indicated that LTX‐315 treatment remarkably reduced the migration and invasion of SKOV3 and A2780 cells compared with the control group (Figure S1C), increased the proportion of apoptotic cells (Figure S1D) and induced cell cycle arrest in the G0/G1 phase (Figure S1E). These data suggested LTX‐315 had tumor‐suppressive effects on OC cells. FIGU RE 1 The establishment of DDP‐resistant OC cells. (A) Wild type SKOV3 and A2780 cells were cultured in Dulbecco's modified Eagle medium containing increasing concentrations of DDP. (B) CCK‐8 assay was utilized to detect the changes of cell viability in each group after the treatment with different concentrations of DDP (SKOV3, A2780, SKOV3/DDP and A2780/DDP), and the IC50 value of DDP was calculated. All the experiments were performed in triplicate. OC, ovarian cancer. ***p < 0.001 3.3 | LTX‐315 reversed the resistance of OC cells to DDP To clarify whether LTX‐315 could reverse DDP resistance of OC cells, SKOV3/DDP and A2780/DDP cells were pretreated with 20 μmol/L LTX‐315 for 48 h. CCK‐8 assay, flow cytometry and Western blot experiments showed that compared with SKOV3 and A2780 cells, in SKOV3/DDP and A2780/DDP cells, the IC50 value of DDP was remarkably higher (Figure 3A,B), the percentage of apoptosis was markedly lower (Figure 3C), and Bax, cleaved caspase 3, and cleaved PARP protein expression levels were weakened, and Bcl‐2 protein expression was augmented (Figure 3D). Compared with drug‐resistant cells treated with 32 μmol/L DDP only, SKOV3/DDP and A2780/DDP cells treated with DDP combined with LTX‐315 showed markedly lower viability (Figure 3A), reduced IC50 of DDP (Figure 3B), remarkably higher percentage of apoptotic cells (Figure 3C), increased expression of Bax, cleaved caspase 3 and cleaved PARP proteins, and decreased expression of Bcl‐2 protein (Figure 3D). These data implied that LTX‐315 treatment markedly reversed the resistance of OC cells to DDP. 3.4 | LTX‐315 exerted its biological effects on OC cells via Beclin‐1/Akt/mTOR signaling pathway To investigate the mechanism of LTX‐315 in reversing DDP re- sistance, the expression levels of Beclin‐1/Akt/mTOR pathway‐related proteins was detected by Western blot. Compared with the control group, Beclin‐1 expression was up‐modulated and both p‐Akt and p‐mTOR expressions were down‐modulated after pretreatment of 20 μmol/L LTX‐315 (Figure 4A). To verify whether the function of LTX‐315 was dependent on the autophagic signaling pathway, OC cells were treated with LTX‐315 (20 µmol/L) + autophagy inhibitor (3‐MA), and the data revealed that compared with the LTX‐315 (20 µmol/L) group, 3‐MA treatment resulted in down‐modulation of Beclin‐1 expression and up‐modulation of both p‐Akt and pmTOR expression (Figure 4A). Next, drug resistance and apoptosis of SKOV3/DDP and A2780/DDP cells, cell viability, apoptosis and apoptosis‐related protein expression levels were detected by CCK‐8 assay, flow cytometry and Western blot, respectively. The data showed that compared with the control group, LTX‐315 treatment (20 μmol/L) group showed reduced cell viability (Figure 4B), decreased IC50 of DDP (Figure 4C), higher apoptotic ratio (Figure 4D), and augmented expression levels of Bax, cleaved caspase 3, and cleaved PARP protein, while Bcl‐2 protein expression was repressed (Figure 4E), importantly, the above effects were reversed after 3‐MA treatment (Figure 4A–E). These data suggested that LTX‐315 prob- ably sensitize OC cells to DDP via regulating Beclin‐1/Akt/mTOR signaling pathway and autophagy. FIGU RE 2 The effects of LTX‐315 on SKOV3/DDP and A2780/DDP cell viability and DDP resistance. (A) Cell counting kit‐8 assay was employed to detect changes in OC cell viability after the treatment with different concentrations of LTX‐315 (control, 10 and 20 μmol/L) for 24, 48, and 72 h, respectively. (B) Based on the above results, the IC50 value of DDP was calculated. All the experiments were performed in triplicate. OC, ovarian cancer. **p < 0.01, and ***p < 0.001. 4 | DISCUSSION At present, DDP‐based chemotherapy is an important strategy for OC treatment. However, DDP resistance seriously affects the prognosis of patients with OC. Currently, there is still a lack of effective methods to reverse chemoresistance of cancer cells in clinical practice.[12–14] En- hancing the sensitivity of OC cells to DDP is particularly critical in the treatment of OC.[15] In this study, we observed that LTX‐315 had the effects of inhibiting the cell viability and promoting the apoptosis in OC cells. Furthermore, LTX‐315 remarkably inhibited the drug resistance of OC cells to DDP. Mechanistically, we reported that LTX‐315 promoted the DDP sensitivity of OC cells through Beclin‐1/PI3K/mTOR signaling pathway. LTX‐315 is a chemically modified 9‐mer cationic peptide, derived from bovine lactoferrin. Previous studies have shown that LTX‐315 is able to transform non‐T cell infiltrating tumors into tumors with antitumor T cell immunity, thus sensitizing unresponsive tumors to immunotherapy, promoting lymphocyte‐independent, and lymphocyte‐ dependent antitumor effects.[16] In addition, in melanoma, LTX‐315 can induce immune cells (such as CD4+ T cells and natural killer cells) to migrate into tumor environment, and this transformation prevents tumor recurrence after treatment and further ensures long‐term antitumor immunity, which makes LTX‐315 a promising antitumor peptide and immunotherapeutic agent.[17] Furthermore, a case report demonstrates that, in a patient with retromammary desmoid tumor infiltrating into the anterior thoracic wall, the intratumoral injection of LTX‐315 blocks the growth of tumor, and the infiltration of CD8 + tumor infiltrating T cell in the lesion is enhanced; meanwhile, the clinical symptoms of the patient are relieved, and the tumor diameter is slightly reduced.[18] This study further implies the promising clinical application foreground of LTX‐315. However, there is few study focusing on whether LTX‐315 can sensitize cancer cells to che- motherapeutics. In the present study, we found for the first time that LTX‐315 could inhibit OC cell viability, as well as promote apoptosis and OC cells' DDP sensitivity, which provided a potential therapeutic approach for the treatment of OC patients, especially those with dis- ease progression and drug resistance. FIGU RE 3 The effects of LTX‐315 in combination with DDP on OC cells. (A,B) Cell counting kit‐8 assay was used to detect the effect of the combination of LTX‐315 and DDP on OC cell viability, and the IC50 of DDP was calculated. (C) Flow cytometry was performed to detect the synergetic effects of LTX‐315 and DDP on the apoptosis of OC cells. (D) Western blot was conducted to detect the synergetic effects of LTX‐315 and DDP on the expression levels of apoptosis‐related proteins. All the experiments were performed in triplicate. GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; PARP, poly (ADP‐ribose) polymerase; OC, ovarian cancer. ***p < 0.001 Accumulating studies have found that PI3K/Akt/mTOR pathway plays a crucial role in the progression of OC.[19–21] PI3K/AKT/mTOR pathway is frequently mutated and activated in OC patients, and it is associated with invasiveness, drug re- sistance, relapse and poor prognosis of the patients.[22] Targeting PI3K/Akt/mTOR signaling is regarded as a potential strategy to treat OC. For instance, bromodomain and extra‐terminal domain (BET) inhibitors (e.g., JQ1) are potentially promising candidates for the treatment of OC, because they can inhibit the prolifera- tion and chemoresistance and promote the apoptosis of OC cells through suppressing Akt/mTOR pathway.[23] Beclin‐1 gene encodes a Bcl‐2‐interacting coiled‐coil protein, which regulates autophagy, and its deletion facilitates the tumorigenesis and cancer progression.[24] Loss of Beclin‐1 expression in OC tissues is a unfavorable prognosticator in patients receiving platinum‐based chemotherapy, and in vitro studies suggest that over-expression of Beclin‐1 reverses the chemoresistance of OC cells to DDP.[25,26] In this study, for the first time, we observed that LTX‐315 promoted Beclin‐1 expression and inhibited p‐PI3k and p‐mTOR expression in OC cells, while 3‐MA could significantly reverse the process. Based on our data, we supposed that LTX‐ 315 could probably inhibit the proliferation of OC cells, promote apoptosis, and sensitize OC cells to DDP through regulating Beclin‐1/PI3K/mTOR signaling and autophagy. In conclusion, our study preliminarily proves that LTX‐315 can kill OC cells and increase their sensitivity to DDP, the pos- sible mechanism of which is to regulate Beclin‐1/PI3K/mTOR signaling pathway. Nonetheless, there are still flaws in this study: first of all, this study is only a preclinical research, with only in vitro data provided. In the following work, in vivo studies are needed to verify our demonstrations. What's more, whether LTX‐315 can sensitize OC cells to other chemotherapeutics by regulating any other signaling pathway is still obscure. Last but not least, as a immunoregulatory peptide, LTX‐315 may synergize with lymphocyte‐mediated antitumor immunity to improve the sensitivity of OC cells to chemotherapeutics, and this scientific hypothesis also remains to be validated in the future. FIGU RE 4 The regulatory effects of LTX‐315 on Beclin‐1/PI3K/mTOR signaling pathway. (A) Western blot was utilized to detect the effects of LTX‐315 and 3‐MA on the expression levels of Beclin‐1, p‐Akt, and p‐mTOR protein in OC cells. (B,C) The effects of LTX‐315 and 3‐MA treatment on OC cell viability were detected by CCK‐8 assay, and the IC50 of DDP was calculated. (D) Flow cytometry was applied to detect the effects of LTX‐315 and 3‐MA on the apoptosis of OC cells. (E) Flow cytometry was adopted to detect the effects of LTX‐315 and 3‐MA on the expression levels of apoptosis‐related proteins in OC cells. All the experiments were performed in triplicate. 3MA, 3‐methyladenine; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; PARP, poly (ADP‐ribose) polymerase; OC,Ruxotemitide ovarian cancer. *p < 0.05, **p < 0.01, and ***p < 0.001