STOML-2 Inhibits Cisplatin-induced Apoptosis In Human Cervical Cancer Cells

BackgroundIn the last decade, gene expression profiling of human cancer has proved valuable in cancer research, providing precious insight into mechanisms and targets involved in oncogenesis in several neoplasms. By microarray analysis of various cancer tissues, we have led to the identification of multiple differentially expressed genes, which maybe serve as more important diagnostic or prognostic markers and even some new treatment targets for cancer.In 2000, the human SLP-2 sequence was first cloned and reported by Wang Y, and it is a novel and unusual member of the stomatin gene superfamily. SLP-2 is tightly associated with the mitochondrial inner membrane but resides within the intermembrane space, between the inner and outer mitochondrial membranes. Moreover, SLP-2 plays a role in regulating the stability of mitochondrial proteins including prohibitins and subunits of respiratory chain complexes.Previous studies have reported that SLP-2 are overexpressed in several types of cancers, such as esophageal squamous cell carcinoma, gallbladder cancer, laryngeal squamous cell carcinoma, breast cancer and gastric cancer. Recent research has shown SLP-2 expression is up-regulated in cervical cancer tissues and significantly related to tumor stage and tumor size. Moreover, patients with high expression of SLP-2 have a significant shorter overall survival and recurrent free survival time compared with those with low SLP-2 expression in cervical cancer. Liyong Zhang et al. have found esophageal squamous cell carcinoma KYSE450 cells transfected with antisense SLP-2 show decreased cell growth, proliferation, tumorigenecity, and cell adhesion. In the current issue of Cancer Biology and Therapy, Wang et al. expand the options for combining metabolic interventions with genotoxic chemotherapeutics by demonstrating that the stomatin-like protein 2 (SLP-2) coordinates bioenergetics and apoptosis. Sandrine Da Cruz’s data show that SLP-2 modulates mitochondrial calcium extrusion, thereby altering the ability of mitochondria to buffer Ca2+ and to shape cytosolic Ca2+ signals. SLP-2 can coordinate apoptosis, however, to the best of our knowledge, SLP-2 gene’s mechanism of inhabiting apoptosis remains less understood.To study the mechanism of action of SLP-2 in apoptosis, we initially use SLP-2 siRNA and Ad-STOML2 to make SLP-2 silenced and overexpressed in human cervical cancer HELA and SIHA cells respectively, and we analyze the effects of SLP-2 protein expression on MEK/ERK signaling pathway and mitochondrial apoptosis pathway after cisplatin are treated for 24 h. Furthermore we find that SLP-2 is upregulated under conditions of high concentrations of cisplatin stress leading to increased protein turnover. Meanwhile, we confirm again that overexpression of SLP-2 can activate of MEK/ERK signaling pathway and suppress of the mitochondrial apoptosis pathway in human cervical cancer cells. These data suggest that SLP-2 can affect the activation of MEK/ERK pathway and inhibit apoptosis by the mitochondrial apoptosis pathway in human cervical cancer cell lines.MethodssiRNA transfection.SLP-2 siRNA duplex were purchased from Invitrogen. To confirm the specificity of the inhibition, the control, nontargeting (NT) siRNA was used as negative control. Cells were transfected with 100 nmol of siRNA duplexes by using Lipofectamine 2000 Reagent according to the manufacturer’s instructions. Knockdown of SLP-2 was quantified by qRT-PCR with GAPDH for normalization after transfection for 24h and by western blotting with α-tubulin for normalization after 72h.Adenovirus infection.Mass production and storage of Ad-STOML2 and Ad-GFP (green fluorescent protein) were purchased from ViGene. For adenovirus infection in HELA cells we used virus diluted to a MOI of 400 pfu/cell and in SIHA cells of 50 pfu/cell. All infected cell cultures were examined for adequate infection efficiency as assessed by western blotting for SLP-2 protein.MTT Assay of Cell ViabilityHELA and SIHA cells after transfection or infection were plated at 0.5× 103cells/well in 96-well plates. Cells were cultured for 72 h and then cell viability was measured by MTT assay according to manufacturer’s instruction. Absorbance at 490 nm was read by using a spectrophotometric plate reader. Each test was performed in triplicate.clonogenic survival assayHELA and SIHA cells were plated on six-well plates at 1000 cells per well for siRNA transfection and at 500 cells per well for adenovirus infection.24 h later, the cells were treated with siRNA transfection and adenovirus infection. Cells were returned to the incubator immediately after treatments to allow colonies to form. After 7 days, the colonies>50 cells were fixed with methanol and stained with crystal violet. Each experiment was repeated at least three times. IC50 (inhibitory concentration 50%) of cisplatin for cellsHELA and SIHA cells were plated at 0.5 × 103 cells/well in 96-well plates for 48 h. Cells were treated with different concentrations of cisplatin, after 24 h using MTT assay to identify their sensitivity to cisplatin and calculate their half inhibition concentration values.Annexin-V/PI double staining assayTransfected HELA or SIHA cells treated with IC50 of cisplatin for 6 h were collected and washed twice with ice-cold PBS and resuspended with 1× binding buffer before incubation with Annexin V-FITC according to the manufacturer’s protocol. After 15 min of incubation at room temperature in the dark, propidium iodide (PI) was added, and the number of stained cells was analyzed using a flow cytometer.Rhod-2 staining assayRhod-2 can combine with calcium ion in the mitochondria specifically, so it is used to determine the level of mitochondrial calcium. Transfected or infected HELA or SIHA cells treated with IC50 of cisplatin for 6 h were stained with dye Rhod-2 according to the manufacturer’s protocol and fluorescent enzyme was used to determinate the relative fluorescence unit(RFU). Ca2+was monitored in these cells by exciting the specimens at 550 nm and observing at 590 nm to detect rhod-2 emission signals.Measurement of mitochondrial membrane potential (MMP)The extent of MMP loss was measured using the potentiometric cation 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1). Transfected HELA or SIHA cells treated with IC50 of cisplatin for 6 h were incubated with JC-1 staining liquid for 20 min at 37℃, washed 3 times with JC-1 staining buffer and examined under flow cytometry. RN A extraction and quantitative real-time PCR (qRT-PCR)Total cellular RNA was extracted from cells using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Total RNA (1μg) was reverse transcribed, and first-strand cDNA was synthesized using reverse transcriptase (Roche, Penzberg, Germany) according to the manufacturer’s instructions. The resulting cDNA was used for quantitative real-time PCR. GAPDH was used as an internal control. qRT-PCR was performed as follows:95℃ for 30 s, one cycle, followed by 95℃ for 5 s and 60℃ for 34 s (40 cycles). After qRT-PCR was finished, dissociation curves were analyzed, and the CT (2-△△CT) method was used to calculate the relative concentration of the amplified products. The experiments were performed for least three times, and the statistical analysis was performed.Western BlottingTransfected or infected HELA or SIHA cells treated with IC50 of cisplatin for 8 h were lysed 30 min with lysis buffer. Following centrifugation at 11000g, supernatants were collected to achieve cytoplasmic proteins and precipitations were collected to achieve mitochondrial proteins. And transfected or infected HELA or SIHA cells treated with IC50 of cisplatin or cells treated with different levels of cisplatin concentration for 24 h were lysed 30 min with lysis buffer to obtain total protein. Following boiling with loading buffer, protein samples were separated by 10% or 12% SDS-PAGE and were transferred onto polyvinylidene difluoride membranes, which were blocked with 5% non-fat milk for 1 h. Subsequently, membranes were incubated with the primary antibodies at 4℃ overnight. Following washing 3 times, the membranes were incubated with the fluorescence-conjugated secondary antibodies at room temperature in the dark for 1 h and visualized with Odyssey IR imaging system. Normalization was ensured by α-tubulin and each band was quantified using Quantity One software.ResultssiRNA transfection and Adenovirus infectionSLP-2 was antagonized by siRNA in HELA and SIHA cells. Hence, the inhibitory effect of SLP-2 were evaluated by qRT-PCR and western blotting and the level of SLP-2 protein expression decreased more than 65% in HELA cells and that of 60% in SIHA cells after 72 hours.SLP-2 was upregulated by Ad-STOML2 in HELA and SIHA cells. Hence, the expression of SLP-2 were evaluated by western blotting and increased more than doubles in HELA cells and that of approximately 3-fold in SIHA cells after 72 hours.SLP-2 enhances proliferation of cervical cancer cell linesTo investigate potential proliferation effects of SLP-2, a colorimetric MTT assay was applied.72 h post-transfection, HELA and SIHA cells, which had been transfected with SLP-2 siRNA, showed a reduced proliferative capacity of 7% and 14%, respectively compared with the cells having been transfected with scrambled siRNA.72 h post-infection, HELA and SIHA cells, which had been infected with Ad-STOML2, showed a increased proliferative capacity of 8% and 5%, respectively compared with the cells having been infected with Ad-GFP.For analysis of long term clonogenic survival, clonogenic survival assay was used. Transfected or infected HELA and SIHA cells demonstrated noticeable differences of viability contrasted with the control groups, which had the same trend with the results of MTT assay.SLP-2 inhibits apoptosis of cervical cancer Cell LinesWe used MTT assay to calculate half inhibition concentration values of cisplatin in HELA and SIHA cells. IC50 of cisplatin for HELA and SIHA cells was approximately 20ug/ml and 15ug/ml.To confirm the apoptotic resistance effect of SLP-2, transfected HELA or SIHA cells treated with IC50 of cisplatin and flow cytometric analysis was performed using annexin V and propidium iodide (PI). We found that Annexin V+/PI-cells (early apoptosis) were more frequently detected in siSLP-2 cells than in scramble cells.SLP-2 is required for the stability of mitochondrial calcium ion and mitochondrial membrane potential (MMP)Calcium ion (Ca2+) is a ubiquitous intracellular signal responsible for numerous cellular events, such as proliferation/growth, differentiation, apoptosis and survival in various cells. Calcium ion overload in the mitochondria is one of the earliest intracellular events that occur following induction of apoptosis. After exposure of HELA and SIHA cells treated with siSLP-2 to IC50 of cisplatin for 6 h, the mitochondrial Ca2+ concentration in HELA cells was increased to 230%±13% of the control value and in SIHA cells was to 192%±18%. The mitochondrial Ca2+ concentration in HELA cells treated with Ad-STOML2 followed by IC50 of cisplatin for 6 h was decreased to 47%k±9% of the control value while in SIHA cells was to 42%±9%.The loss of mitochondrial membrane potential is an early event in cell apoptosis and results from the reduction of the electrochemical gradient of mitochondrial membrane. To investigate whether the apoptotic resistance effect of SLP-2 in HELA and SIHA cells involves alterations of MMP, we used a JC-1 probe to monitor the change of MMP by flow cytometry. After exposure to IC50 of cisplatin for 6 h, the MMP of HELA and SIHA cells treated with siSLP-2 was significantly increased relative to that of control (3.8%vs17.9%,3.2%vs25.1%).The data suggested that mitochondrial dysfunction might be involved in the process of IC50 of cisplatin-induced apoptosis in HELA and SIHA cells because of the change of SLP-2 expression.Effects of SLP-2 on expressions of p-MEK1/2, p-ERK1/2, Bax, Bcl-2, caspase 3, cleaved-caspase 3, PARP, cleaved-PARP proteins and cytochrome C release from mitochondria into cytosolCalcium channel has a very important influence on activation of the RAF-MEK-ERK mitogen-activated protein kinase pathway, therefore, we next examined the effect of SLP-2 on the activities of p-MEK1/2 and p-ERK1/2. We found that the expression level of p-MEK1/2 and p-ERK1/2 had the same trend with the expression level of SLP-2 in HELA and SIHA cells.Bcl-2 and Bax are signaling molecules involved in the mitochondrial pathway of caspase 3-dependent apoptosis, which is associated with the release of cytochrome C (cyt-C) from the mitochondrial matrix to the cytoplasm. We found a decrease of the ratio of Bax/Bcl-2, cleaved-caspase 3/caspase 3 and cleaved-PARP/PARP in high expression of SLP-2 group of HELA and SIHA cells and decreased expression of cytochrome C release from mitochondria into cytosol was also observed.Effects of different concentrations of cisplatin on expressions of SLP-2, p-MEK1/2, p-ERK1/2, Bax, Bcl-2, caspase 3, cleaved-caspase 3, PARP, cleaved-PARP proteinsWhen HELA cells were treated with different levels of cisplatin concentration (0, 1×IC50,2×IC50,3×IC50,4×IC50, respectively) for 8 h or 24 h, we found that the increase of expression of SLP-2 along with the increase of the concentration of cisplatin by qRT-PCR and western blotting.To further investigate the effect of SLP-2 on MEK/ERK signaling pathway and the mitochondrial apoptosis pathway, different levels of cisplatin concentration (0, 0.5xIC50, 1×IC50,1.5×IC50,2×IC50,respectively) were used to treat HELA and SIHA cells for 24 h to test expressions of SLP-2, p-MEK1/2, p-ERK1/2, Bax, Bc1-2, caspase 3, cleaved-caspase 3, PARP, cleaved-PARP proteins. We found that the expression of SLP-2 in cells treated with 1 xIC50 of cisplatin was elevated slightly compared with non-cisplatin group and in cells treated with 2xIC50 of cisplatin was markedly higher than the 1 xIC50 of cisplatin group which was tested by qRT-PCR and western blotting. Furthermore, an increase of the expression level of p-MEK1/2 and p-ERKl/2 and a decrease of the ratio of Bax/Bcl-2, cleaved-caspase 3/caspase 3 and cleaved-PARP/PARP were observed in 2xIC50 of cisplatin group compared with 1×IC50 of cisplatin group.Conclusions1. The overexpression of STOML-2 enchances the viability of human cervical cancer cells.2. The overexpression of STOML-2 inhibits the apoptosis of human cervical cancer cells.3. Our findings reveal for the first time that STOML-2 inhibits the apoptosis by the activation of MEK/ERK signaling pathway and the suppression of the mitochondrial apoptosis pathway in human cervical cancer cells.4. The expression of STOML-2 is up-regulated under stress of high cisplatin concentrations in human cervical cancer cells.