Contributed equally
Cervical cancer is one of the primary causes of cancer-associated mortality worldwide. Due to the increasing incidence of cervical cancer, multiple treatment options are required. Initial responses to chemotherapy and surgical interventions are generally positive, however patients often experience relapse and tumor recurrence. Currently, the effects of cucurbitacins on different types of cancer are being investigated, as they exhibit a wide variety of bioactivities. The anticancer activity of the cucurbitacin 23,24-dihydrocucurbitacin B against a panel of human cervical cancer cell lines was investigated in the current study. Cell viability was determined using an MTT assay and apoptosis was detected using DAPI staining. The proportion of apoptotic cells, cell cycle distribution, mitochondrial membrane potential (ΔΨm) and reactive oxygen species (ROS) levels were estimated using flow cytometry. Protein expression was determined using western blot analysis. The results of the current study indicated that 23,24-dihydrocucurbitacin B inhibited the viability of human cervical cancer cell lines and had an IC50 of 40–60 µM. However, its cytotoxic effects were much less pronounced in normal epithelial fr2 and HerEpiC cells, where it had an IC50 of 125 µM. The underlying mechanisms of this were further studied and the results demonstrated that 23,24-dihydrocucurbitacin B induced apoptosis in HeLa cells and caused ROS-mediated shifts in the ΔΨm. Additionally, it caused the cell cycle arrest of HeLa cells at the G2/M checkpoint. The phosphoinositide 3 kinase/protein kinase B/mechanistic target of rampamycin (PI3K/AKT/mTOR) cascade may serve an important role in cancer tumorigenesis, progression and resistance to chemotherapy. The results indicated that 23,24-dihydrocucurbitacin B significantly decreased the expression of important proteins in the PI3K/Akt/mTOR cascade. Taken together, these results suggest that 23,24-dihydrocucurbitacin B may be novel method of treating cervical cancer.
Cucurbitacins are secondary plant metabolites chemically categorized as steroids. They are synthesized by a range of plant species, particularly by those that are part of the cucurbitaceae family (
The present study evaluated the effect of the cucurbitacin 23,24-dihydrocucurbitacin B on human cervical cancer cells. Its underlying mechanism of action was assessed with particular emphasis on the effect of 23,24-dihydrocucurbitacin B on the phosphoinositide 3 kinase/protein kinase B/mechanistic target of rampamycin (PI3K/Akt/mTOR) cascade. The expression of proteins in the PI3K/Akt/mTOR pathway is dysregulated in several types of cancer (
The cervical cancer cell lines C33A, ME-180, C4-1 and HeLa, the normal cell line fR2 and human cervical epithelial cells (HCerEpiC) were obtained from the Cancer Research Institute of Beijing (Beijing, China) and maintained in Dulbecco's modified Eagle's medium (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Invitrogen; Thermo Fisher Scientific, Inc.), 100 µg/ml streptomycin and 100 U/ml penicillin G (HiMedia, West Chester, Pennsylvania, USA) in an incubator at 37°C with 5% CO2.
The effect of 23,24-dihydrocucurbitacin B (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) on the viability of different cervical cancer cell lines and normal fR2 and HCerEpiC cells was evaluated using an MTT assay. Cells were seeded at 1×106 cells/well in 96-well plates for 12 h and then treated with different concentrations of 23,24-dihydrocucurbitacin B (0, 0.78, 1.56, 3.12, 6.25, 12.5, 25, 50, 100 and 200 µM) for 24 h. A total of 20 µl MTT solution (2.5 mg/ml) for 24 h was then added to each well. The medium was removed and 500 µl dimethyl sulfoxide was added to each well to dissolve formazan crystals. Optical density was recorded using an ELISA plate reader at a wavelength of 570 nm. 23,24-dihydrocucurbitacin B exhibited marked anticancer activity against all cell lines; however, further experiments were performed on the HeLa cancer cell line alone as the lowest MIC was observed against this cell line.
HeLa cells were cultured to the exponential phase (70% confluence), collected and counted using a hemocytometer. Cells were then seeded at a density of 200 cells/well and incubated for 24 h to allow cells to adhere. Cells were then treated with different concentrations of 23,24-dihydrocucurbitacin B (0, 20, 40 and 80 µM). Cells were incubated for 6 days and then washed with PBS. This was followed by fixation with 70% methanol at −20°C for 24 h and staining with 0.01% (w/v) crystal violet for 35 min at 25°C. Cells were then counted in 10 fields using a light microscope at a magnification of ×200.
HeLa cells were cultured to a density of 2×105 cells/well in 6-well plates and were subsequently treated with 0, 20, 40 and 80 µM 23,24-dihydrocucurbitacin B for 24 h. Cells were then stained with DAPI for 20 min at room temperature. The cells were then fixed with 70% methanol at −20°C overnight and observed using fluorescence microscopy (magnification, ×200). A similar procedure was followed for Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) (Sigma-Aldrich; Merck KGaA) staining; cells were stained with annexin V/PI and investigated using a flow cytometer, (BD Biosciences, San Jose, CA, USA) following the manufacturer's protocol and BD FACSuite software version 1.0 for analysis.
HeLa cells were seeded at a density of 2×105 cells/well in 6-well plates and incubated for 24 h. Cells were then treated with 0, 20, 40 and 80 µM 23,24-dihydrocucurbitacin B for 24 h at 37°C in 5% CO2. Cells were washed twice with PBS and resuspended in 500 µl dihydrofluorescein diacetate (10 µM) (Sigma-Aldrich; Merck KGaA) for mitochondrial ROS estimation and DiOC6 (1 µmol/l) at 37°C in a dark room for 35 min to measure the ΔΨm. Samples were then investigated using a flow cytometer following a previously described protocol (
HeLa cells were harvested and washed twice with PBS. Cells were then fixed with 70% ethanol for ~1 h at −20°C and then washed again with PBS. Cells were resuspended in a solution of PI (50 µl/ml) and RNase1 (250 µg/ml) (Invitrogen; Thermo Fisher Scientific, Inc.). This was followed by incubation for 30 min at room temperature and fluorescence-activated cell sorting using 10,000 cells/group with a flow cytometer.
Following treatment with various concentrations of 23,24-dihydrocucurbitacin B, cells were harvested and lysed in radioimmunoprecipitation lysis buffer (20 mM HEPES, 350 mM NaCl, 20% glycerol, 1% Nonidet P 40, 1 mM MgCl2, 0.5 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, 1 mM, phenylmethane sulfonyl fluoride, protease inhibitor cocktail and phosphatase inhibitor cocktail). The protein concentration was determined by BCA assay. A total of 20 µg protein/lane was separated on 10% SDS-PAGE gel. Proteins were then transferred to nitrocellulose membranes, blocked with 5% bovine serum albumin (Invitrogen; Thermo Fisher Scientific, Inc.), for 45 min at room temperature and probed with the following primary antibodies overnight at 4°C: Actin (cat. no. sc-58673), Akt (cat. no. sc-135829), phosphorylated (p)-AKT (cat. no. sc-7985-R), P13K (cat. no. sc-136298), p-P13K (cat. no. sc-100407), mTOR (cat. no. sc-517464) and p-mTOR (cat. no. sc-293133; all 1:1,000). All antibodies were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Proteins were then incubated with horseradish peroxidase-conjugated anti-rabbit secondary antibody (cat. no. sc-2357-CM) for 1 h overnight at 4°C. WEST-SAVE Up™ luminal-based enhanced chemiluminescent reagent was then used to visualize bands (ABFrontier, Co., Ltd., Seoul, Korea).
Experiments were performed in triplicate and data are presented as the mean ± standard deviation. Statistical analysis was performed by GraphPad prism 7 (GraphPad Software, Inc., La Jolla, CA, USA). Student's t test was used for comparison between 2 samples and one way analysis of variance followed by a Tukey's post hoc test was used for comparisons between >2 samples. P<0.01 was determined to indicate a statistically significant difference.
The cytotoxic potential of 23,24-dihydrocucurbitacin B (
DAPI staining was performed to investigate whether 23,24-dihydrocucurbitacin B exerts antiproliferative effects on HeLa cells by inducing apoptosis. The results of DAPI staining indicated that 23,24-dihydrocucurbitacin B caused marked apoptosis in HeLa cells (
The pro-apoptotic potential of 23,24-dihydrocucurbitacin B observed following DAPI staining suggested that it may cause the accumulation of intracellular ROS. Therefore ROS levels were estimated in HeLa cells treated with different doses of 23,24-dihydrocucurbitacin B for 24 h. The results indicated that the intracellular ROS levels of treated cells significantly increased, by 70–260% compared with untreated cells (
ROS generation is associated with mitochondrial dysfunction, as it disturbs the outer mitochondrial potential in order to discharge apoptosis-promoting proteins (
Cell cycle arrest is one of the important mechanisms by which anticancer agents exert their inhibitory effects (
The effect of 23,24-dihydrocucurbitacin B on the expression of some of the proteins involved in the mTOR/PI3K/Akt cascade was evaluated using western blotting. The results indicated that the expression of mTOR, p-mTOR, PI3K, p-PI3K and p-Akt was markedly decreased in HeLa cells following treatment with 23,24-dihydrocucurbitacin B (
Cervical cancer is one of the most common types of cancer diagnosed in women worldwide and ~5 Lakh women are diagnosed with the disease annually (
It has also been demonstrated that various anticancer drugs, including cisplatin, Taxon and 5-fluorouracil (
In addition, the results of the current study indicated that 23,24-dihydrocucurbitacin B-treated cells exhibited a reduction in the ΔΨm, which was mediated by ROS. These results are consistent with those of a previous study (
Flow cytometric analysis in the current study indicated that 23,24-dihydrocucurbitacin B induced G2/M cell cycle arrest and markedly increased the proportion of HeLa cells in the G2 phase in a dose-dependent manner. These results are consistent with those of a previous study, which demonstrated that 23,24-dihydrocucurbitacin B induces G2/M cell cycle arrest in breast cancer cells (
The PI3K/Akt/mTOR signaling pathway is considered to be an important target for anticancer chemotherapy (
In conclusion, the results of the current study indicate that 23,24-dihydrocucurbitacin B may be a potential candidate for the management of cervical cancer by inducing apoptosis, cell cycle arrest and regulating the mTOR/PI3K/Akt signaling pathway. There are limited effective treatments available for cervical cancer; the low toxicity associated with the naturally occurring 23,24-dihydrocucurbitacin B means that it may be developed as a novel treatment for cervical cancer. However, further studies are required to validate its effectiveness in cervical cancer.
The effect of 23,24-dihydrocucurbitacin B on the HeLa cervical cancer cell line. (A) Chemical structure of 23,24-dihydrocucurbitacin B. (B) Effect of 0, 20, 40 and 80 µM 23,24-dihydrocucurbitacin B on the viability of HeLa cervical cancer cells. (C) Effect of 0, 20, 40 and 80 µM 23,24-dihydrocucurbitacin B on the morphology of HeLa cervical cancer cells. Magnification, ×200. All experiments were performed in triplicate and values are expressed as the mean ± standard deviation. The differences between the two cell lines at indicated concentrations were considered significant at *P<0.01, **P<0.001 and ***P<0.0001 at different doses between the two cell lines (HeLa vs. HCerEPiC).
Effect of 23,24-dihydrocucurbitacin B on colony formation in HeLa cells. (A) Images of the colony formation potential of HeLa cells treated with 0, 20, 40 and 80 µM 23,24-dihydrocucurbitacin B. (B) Quantification of colonies of HeLa cells at the indicated doses. All results are representative of three biological replicates and values are expressed as the mean ± standard deviation. *P<0.01, **P<0.001 and ***P<0.0001 vs. untreated cells.
DAPI staining indicating the induction of apoptosis in HeLa cells treated with 0, 20, 40 and 80 µM 23,24-dihydrocucurbitacin B. All results are representative of three biological experiments. Red arrows indicate apoptotic cells. Magnification, ×200.
Estimation of apoptotic population of HeLa cells treated with 0, 20, 40 and 80 µM 23,24-dihydrocucurbitacin B using Annexin V-FITC/PI staining and flow cytometry. (A) Flow cytometric analysis. (B) The apoptosis of HeLa cells. All results are representative of three biological experiments. All experiments were carried out in triplicate and values are expressed as the mean ± standard deviation. *P<0.01, **P<0.001 and ***P<0.0001 vs. untreated cells. PI, propidium iodide; FITC, fluorescein isothiocyanate.
Effect of 0, 20, 40 and 80 µM 23,24-dihydrocucurbitacin B on ROS generation and ΔΨ
Effect of 0, 20, 40 and 80 µM 23,24-dihydrocucurbitacin B on the cell cycle distribution of HeLa cells. All results are representative of three biological experiments.
Effect of 0, 20, 40 and 80 µM 23,24-dihydrocucurbitacin B on the expression of mTOR/PI3K/Akt signaling pathway proteins in HeLa cells. All results are representative of three biological experiments. mTOR, mechanistic target of rampamycin; PI3K, phosphoinositol 3 kinase; Akt, protein kinase B; p-, phosphorylated.
IC50 of 23,24-dihydrocucurbitacin B against different cervical cancer and normal cell lines as determined by MTT assay.
Cell line | IC50 (µM) |
---|---|
C33A | 60 |
ME-180 | 50 |
C4-1 | 40 |
HeLa | 40 |
FR2 | 125 |
HCerEpiC | 125 |
IC50, half maximal inhibitory concentration.