Epithelial-mesenchymal transition-related signaling pathways in gastric Cancer cells distinctively respond to long-term experimental ketosis.

BACKGROUND: The interrelationship between cellular metabolism and the epithelial-to-mesenchymal transition (EMT) process has made it an interesting topic to investigate the adjuvant effect of therapeutic diets in the treatment of cancers. However, the findings are controversial. In this study, the effects of glucose limitation along and with the addition of beta-hydroxybutyrate (bHB) were examined on the expression of specific genes and proteins of EMT, Wnt, Hedgehog, and Hippo signaling pathways, and also on cellular behavior of gastric cancer stem-like (MKN-45) and non-stem-like (KATO III) cells. METHODS AND RESULTS: The expression levels of chosen genes and proteins studied in cancer cells gradually adopted a low-glucose condition of one-fourth, along and with the addition of bHB, and compared to the unconditioned control cells. The long-term switching of the metabolic fuels successfully altered the expression profiles and behaviors of both gastric cancer cells. However, the results for some changes were the opposite. Glucose limitation along and with the addition of bHB reduced the CD44+ population in MKN-45 cells. In KATO III cells, glucose restriction increased the CD44+ population. Glucose deprivation alleviated EMT-related signaling pathways in MKN-45 cells but stimulated EMT in KATO III cells. Interestingly, bHB enrichment reduced the beneficial effect of glucose starvation in MKN-45 cells, but also alleviated the adverse effects of glucose restriction in KATO III cells. CONCLUSIONS: The findings of this research clearly showed that some controversial results in clinical trials for ketogenic diet in cancer patients stemmed from the different signaling responses of various cells to the metabolic changes in a heterogeneous cancer mass.

Inhibition of MiR-155 Using Exosomal Delivery of Antagomir Can Up-Regulate PTEN in Triple Negative Breast Cancer.

BACKGROUND: The most aggressive form of breast cancer (BC) is Triple-Negative BC (TNBC), with the poorest prognosis, accounting for nearly 15% of all cases. Since there is no effective treatment, novel strategies, especially targeted therapies, are essential to treat TNBC. Exosomes are nano-sized microvesicles derived from cells and transport various intracellular cargoes, including microRNAs (miRNAs). MiRNAs, small non-coding RNA, are an influential factor in the development of cancerous transformations in cells. METHOD: Bioinformatics analysis of genes related to TNBC revealed that PTEN plays a crucial role in the disease. Relative expression of this gene was analyzed with RT-qPCR in 14 TNBC clinical samples. Electroporation was used to load miRNA antagomir into exosomes extracted from the conditioned medium. Then, the expression of miR-155 and PTEN was evaluated in MDA-MB-231 cells treated with antagomir-loaded exosomes. RESULTS: Based on the bioinformatics analysis, miR-155 is a potent inhibitor of PTEN. Following treatment with antagomir-loaded exosomes, RT-qPCR showed significantly reduced miR- 155 and increased PTEN levels in MDA-MB-231 cells. CONCLUSION: Based on the results of this study, exosomes can be effectively used as a cargo of oligonucleotides like miRNA mimics and antagomirs in targeted therapies.

Comparison of DPPIV Levels in Serum and Tumour of OSCC Patients and Its Correlation with Active Matrix Metalloproteinases 2 and 9.

INTRODUCTION: The important role of Dipeptidyl Peptidase IV (DPPIV) has been reported in tumour progression of several human cancers. This study demonstrates the DPPIV mRNA expression level and activity in tumour and paired non-tumour tissues of oral squamous cell carcinoma (OSCC) patients and the potential modulation of DPPIV in the metastasis of tumour through regulating MMP2 and MMP9 activities. MATERIALS AND METHODS: This study was conducted on 16 OSCC patients. The mRNA expression level of DPPIV was evaluated by RT-qPCR in tumour of OSCC patientsand compared with their paired non-tumour tissues. Additionally, DPPIV activity was measured in serum, tumour and paired non-tumour tissues of OSCC patients. Zymography was performed to measure and compare the activities of MMP2 and MMP9 between tumour and paired non-tumour tissues of OSCC patients. RESULTS: The results showed significantly higher DPPIV mRNA level and activity in tumour of OSCC patients compared to their paired non-tumour tissues. Tumour DPPIV mRNA expression and activity were positively correlated with activities of MMP2 and MMP9, respectively. Serum DPPIV activity of OSCC patients was lower compared to healthy control and did not show correlation with tumour DPPIV mRNA level. CONCLUSION: These data indicate that secreted DPPIV may not originate from the tumour tissue of OSCC patients. Furthermore, increased DPPIV gene expression and activity in tumour of OSCC patients might be involved in the ECM degradation and invasion of OSCC through regulation of MMP2 and MMP9 activities.

Downregulation of NRARP exerts anti-tumor activities in the breast tumor cells depending on Wnt/ß-catenin-mediated signals: The role of miR-130a-3p.

The Notch-regulated ankyrin repeat protein (NRARP) functions as a molecular link between Notch and Wnt signaling pathways. Although it has recently been identified to be overexpressed in breast cancer (BC), the molecular mechanisms that regulate NRARP remain unknown. Since microRNAs (miRNAs) regulate gene expression post-transcriptionally, miRNA dysregulation could explain the abnormal gene expression. Here, we identified miR-130a-3p as an NRARP regulator and evaluated its effects on the behavior of BC cells. Quantitative real-time PCR was performed to assess the transcriptional levels of miR-130a-3p and NRARP in BC cells. Next, miR-130a-3p was transiently transfected into BC cells to assess its influence on NRARP expression. Owing to the positive regulatory effects of NRARP on the Wnt/ß-catenin signaling pathway, we also analyzed the expression levels of five Wnt/ß-catenin pathway genes and one downstream target gene in BC cells. We then assessed anti-tumor activities of miR-130a-3p in BC cells using the MTT proliferation assay, the soft agar colony formation assay for anchorage-independent growth (AIG), as well as scratch and transwell assays for cell migration. The results showed that miR-130a-3p was downregulated in BC cells, whereas NRARP was upregulated. Overexpression of miR-130a-3p inhibited the expression of NRARP and some Wnt/ß-catenin signaling pathway genes, as well as exerted anti-tumor effects as evidenced by decreased cell proliferation, AIG, and migration of BC cells. In conclusion, the tumor-suppressive function of miR-130a-3p in BC may be mediated by inhibiting NRARP and Wnt/ß-catenin signaling pathway. As a result, miR-130a-3p could be introduced as a therapeutic target for miRNA therapy in BC.

The effect of ghrelin on antioxidant status in the rat's model of Alzheimer's disease induced by amyloid-beta.

Alzheimer's disease (AD) is a neurodegenerative disorder associated with amyloid-beta (Aß) plaque formation and oxidative stress in the brain. Ghrelin has been proven to exert antioxidant activity and neuroprotection in different neurological diseases. This study is going on to examine the effect of ghrelin on antioxidant status in the rat's model of AD induced by Aß. Cognitive impairment was induced by intra-hippocampal administration of Aß (10 µg) in Wistar rats and ghrelin (80 µg/kg) was administrated intraperitoneal for ten consecutive days. Behavior was assessed with Morris water maze and passive avoidance tests. Malondialdehyde (MDA) level as a marker of lipid peroxidation was assessed using the thiobarbituric acid. Catalase activity was assayed by the decomposition of H2O2. Antioxidant capacity was determined using the FRAP method. Treatment with ghrelin decreased the hippocampus and serum MDA levels in wild-type rodents and prevented an increase in hippocampal and serum MDA levels in animals receiving Aß. There was no significant change in the serum catalase activity between the studied groups. Hippocampus catalase activity was reduced in the Aß group and treatment with ghrelin increased it. The antioxidant capacity of the hippocampus and serum increased in the ghrelin-receiving control group. The hippocampus antioxidant capacity level decreased in the Aß group, and treatment with ghrelin increased it, but there were no significant changes in the serum antioxidant capacity of animals receiving Aß. These results provide evidence that the administration of ghrelin has antioxidant properties and protects against hippocampal lipid peroxidation in a rat model of AD.

Long-Term Glucose Restriction with or without ß-Hydroxybutyrate Enrichment Distinctively Alters Epithelial-Mesenchymal Transition-Related Signalings in Ovarian Cancer Cells.

The beneficial impacts of the ketogenic diet and metabolic reprograming were recently reported for ovarian cancer patients. In this study, the effects of glucose restriction with or without beta-hydroxybutyrate (bHB) enrichment were studied in drug-resistant CD133high A2780CP and CD133low SK-OV-3 ovarian cancer cells to scrutinize the impact of experimental ketosis on ATP production, epithelial to mesenchymal transition (EMT), and related signaling pathways including Wnt, Hippo, and Hedgehog. Cells were adapted and maintained for a month with restricted levels of glucose (250 mg/l) with or without the therapeutic concentration of bHB (5 mM). Quantitative PCR, Western blot analysis, flow cytometry, chemiluminescence, and wound healing assay were used in this study. Glucose restriction and bHB enrichment reduced the stemness marker and diminished In Vitro migration in both cell lines. Glucose restriction significantly reduced ATP levels in both cells, but bHB enrichment was partially compensated for the ATP levels solely in SK-OV-3 cells. Glucose restriction mainly inhibited the Wnt pathway in the CD133high A2780CP cells, but the Hedgehog pathway was the main target in CD133low SK-OV-3 cells. In Conclusion, Prior targeted evaluations of key genes' expression would help to predict the distinctive impacts of metabolic fuels and to optimize the efficacy of ketogenic diets.

MiR-130a-3p blocks Wnt signaling cascade in the triple-negative breast cancer by targeting the key players at multiple points.

OBJECTIVES: Aberrant Wnt signaling cascade is a hallmark of the triple-negative breast cancer (TNBC) that is linked with the increased proliferation, invasion, and poor overall survival. many genes are post-transcriptionally regulated by microRNAs (miRNAs) therefore; it is indisputable that the dysregulation of the miRNAs is an explanation for the aberrant signaling cascades. Thus, the present study was conducted to find the putative miRNA targeting the key players of Wnt/ß -catenin cascade in the TNBC. METHODS: The miR-130a-3p was found as a potential regulator of the Wnt signaling cascade by applying several bioinformatic algorithms. Quantitative real-time PCR (qRT-PCR) was used to analyze the expression levels of miR-130a-3p and Wnt cascade genes in the TNBC cells. Afterward, TNBC cells were transiently transfected with the miR-130a-3p to investigate its effects on the expression of Wnt cascade genes. Subsequently, MTT, soft agar colony formation, scratch, transwell cell migration, and transwell cell invasion assays were used to determine the behavior of the TNBC cells in response to miR-130a-3p restoration. RESULTS: Results of the qRT-PCR showed downregulation of miR-130a-3p and upregulation of the Wnt cascade genes in the TNBC cells compared to the normal cells. Transient overexpression of miR-130a-3p decreased the expression levels of Wnt cascade genes significantly in the TNBC cells. Moreover, following the miR-130a-3p overexpression, the proliferation, anchorage-independent growth, and migration of the TNBC cells were reduced. CONCLUSION: Overall, our findings provided an evidence for the significant role of miR-130a-3p in the regulation of Wnt/ß-catenin cascade, and also introduced the miR-130a-3p as a new therapeutic target for the patients with TNBC.

Effects of Quinacrine on Expression of Hippo signaling Pathway Components (LATS1, LATS2, and YAP) in Human Breast Cancer Stem Cells.

OBJECTIVE: The Hippo signaling pathway has important role in the pathogenesis of some tumors. Breast cancer is the most prevalent cancer among females in the world. In recent years, various articles referred to inhibiting effect of quinacrine, a derivative of 9-aminoacridine, on the growth of several types of cancer cells. In this study, we evaluated the effect of quinacrine on expression of LATS1, LATS2, and YAP genes of the Hippo signaling pathway and YAP level in human breast cancer stem cells (MDA-MB 231 cell line). This cell line of breast cancer expresses the triple negative characteristics. METHODS: MDA-MB 231 cells was treated with 0.5 µM of quinacrine for 3 days. The dose was selected using MTT assays. The expression of genes was quantified by Real-time PCR. The protein expression was performed by Western blotting. Significance of observations were checked by means of Mann-Whitney test using p.

Evaluation of Impacts of Cellular Metabolism on the Migration of Ovarian Cancer Cells by Two in Vitro Assays: A Method Comparison Study.

BACKGROUND: Alteration of metabolic pathways in cancer cells can intensely modulate their migration as an important step in invasion and metastasis. Ketogenic diet showed some contradictory results in cancer patients. In this study the impact of metabolic reprogramming of A2780CP as a model of ovarian cancer stem-like cells on cell migration by two in vitro methods: wound healing and soft agar colony-forming assays. MATERIALS AND METHODS: short term and long term metabolic reprogramming were done by restriction of glucose to 250mg/L with or without enrichment with beta-hydroxybutyrate (5 milimolar) for 48 hours and 30 days, respectively. Wound healing assay was done and the wound ratio was calculated for 24 and 48 hours. Soft agar colony formation assay was also done in treated and control cells. For method comparison, ten biological replicates were analyzed in triplicate. RESULTS: Migration of A2780CP ovarian cancer stem-like cells were significantly alleviated by long term glucose restriction but no significant changes were observed in short term study. Beta-hydroxybutyrate enrichment did not produce significant impacts on glucose restriction in short or long term studies. CONCLUSION: The results of colony formation in soft agar and wound or scratch healing assay were in good correlation and convergence which could be used interchangeably in the investigation of metabolic reprogramming in cancer cells.

Epigallocatechin-3-gallate Enhances the Efficacy of MicroRNA-34a Mimic and MicroRNA-93 Inhibitor Co-transfection in Prostate Cancer Cell Line.

The potential role of microRNAs (miRNA or MIR) as therapeutic molecules has moved them from basic research to the field of cancer therapy. High expression of miR-93 and low expression of miR-34a have previously been indicated in prostate cancer (PC), which is the second leading cause of cancer-related death in men. Androgen receptor (AR) and prostate-specific antigen (PSA) play key roles in the initiation and progression of this cancer. Therefore, this study aimed to investigate the effects of the transfection and co-transfection of miR-34a mimic and miR-93 inhibitor with or without epigallocatechin-3-gallate (EGCG) on prostate cancer cell line and also to evaluate their effects on the expression of AR, PSA. Human lymph node carcinoma of the prostate (LNCaP) cells were treated with miR-34a mimic or/and miR-93 inhibitor with or without EGCG. Gene or protein expressions were assessed by real-time PCR or western blotting of lysates. The transfection with miR-34a mimics significantly reduced the mRNA expression of AR (p=0.0016), and PSA (p=0.038) compared to the control. Also, the miR-93 inhibitor led to a decrease in the mRNA expression of AR (p=0.0057) and PSA (p>0.05) compared to the control group. Furthermore, the co-transfection, along with EGCG, caused more decrease in both the AR (p<0.001) and the PSA (p=0.003) expression compared with the co-transfection without EGCG. Our study indicates that the reduced expression of AR and PSA in PC cells followed by treatment with miR-34a mimic and miR-93 inhibitor and their combination with EGCG as a natural substance may be a promising therapeutic way for controlling the growth of these malignant cells.

Histone Butyrylation/ Acetylation Remains Unchanged in Triple Negative Breast Cancer Cells after a Long Term Metabolic Reprogramming.

BACKGROUND: Altered metabolism is one of the hallmarks of the cancer cells which reciprocally interrelate with epigenetic processes, such as post-translational histone modifications to maintain their desired gene expression profiles. The role of beta-hydroxybutyrate as a ketone body in cancer cell biology and histone modifications are reported. The present study aimed to evaluate the impacts of long-term metabolic reprogramming via glucose restriction and beta-hydroxybutyrate treatment on histone acetylation and butyrylation in MDA-MB231 cells as a model of triple negative stem-like breast cancer. METHODS: For long-term treatment, cells were set up in three groups receiving DMEM with restricted glucose (250 mg/L), DMEM with restricted glucose but enriched with five millimolar beta-hydroxybutyrate and DMEM with standard glucose (1gL) and investigated for a month. Histone modifications, including H3 acetylation and butyrylation, were investigated by immunoblotting after an acid extraction of the histone proteins. RESULTS AND CONCLUSION: Neither beta-hydroxybutyrate enrichment nor glucose restriction elicited a significant effect on the butyrylation or acetylation level of histone H3 upon a long-term treatment. Metabolic plasticity of cancer cells, mainly stem-like triple negative breast cancer cells alleviate or neutralize the impact of long-term metabolic reprogramming via restriction of glucose and histone modifications enrichment. These results shed new light upon the mechanism of controversial efficacy of ketogenic diets in clinical trials.

The relationship between 174 G/C and -572 G/C of IL-6 gene polymorphisms and susceptibility of celiac disease in the Iranian population.

INTRODUCTION: Celiac disease (CD) is a chronic inflammatory intestinal disorder. Different immunological factors, including inflammatory cytokines, may play an important role in disease susceptibility. AIM: To investigate the relationship between -174G/C and -572G/C gene polymorphisms and the serum level of interleukin 6 (IL-6) and susceptibility to CD in the Iranian population. MATERIAL AND METHODS: In this case-control study blood samples were collected of 105 patients with CD and 106 healthy subjects randomly in 2016 and evaluated by polymerase chain reaction-restriction fragments length polymorphism (PCR-RFLP) method. A sequence was also used to confirm the results of both polymorphisms. The IL-6 concentration was measured using ELISA. RESULTS: The results showed a significant relationship between polymorphism -572G in CD patients when compared with control subjects by genotype (p = 0.001) and alleles (p = 0.022), respectively. There was no significant relationship between polymorphism 174G and frequency of genotype, but an association of this polymorphism with the frequency of alleles (p = 0.034), age (p = 0.001), and body mass index (p = 0.003) was seen. The serum level of interleukin-6 was significantly associated only with rs1800796 (p < 0.001). CONCLUSIONS: The results confirm previous studies in different parts of the world and indicate that IL-6 (572G/C) polymorphism may play a role in susceptibility to CD in the Iranian population.

Structural characterization of recombinant human fibroblast growth factor receptor 2b kinase domain upon interaction with omega fatty acids.

The mutated recombinant kinase domain of human fibroblast growth factor receptor 2b (hFGFR2b) is overexpressed and purified, and its structural changes upon the interaction with three unsaturated fatty acids (UFAs), oleic, linoleic and α-linolenic are studied. This interaction is investigated to find out about the folding and unfolding effect of unsaturated fatty acids on the kinase domain structure of hFGFR2b. Recombinant pLEICS-01 vectors, containing the mutated coding region of hFGFR2b, are expressed in the standard Escherichia coli BL21 (DE3) host cells and purified by Ni2+-NTA affinity chromatography. While polyacrylamide gel electrophoresis characterizes the functionality of recombinant protein, its structural changes are studied in the presence and absence of various concentrations of oleic, α-linolenic and linoleic acids using circular dichroism (CD) and fluorescence spectroscopy. Far ultraviolet CD results show that unsaturated fatty acids do not change the secondary structure of the recombinant kinase domain of hFGFR2b. However, chemical denaturation analysis confirms that all three UFAs destabilize the tertiary structure of recombinant protein. A decrease in the fluorescence intensity without any significant red or blue shift (336±1nm) reflects a variation in the tertiary structure of protein. The direct interaction of the studied UFAs with hFGFR2b reduces the conformational stability of their kinase domains. The structural changes in hFGFR2b in the presence of UFAs may be necessary for hFGFR2b to adjust the signal transduction and regulate the key cellular processes.

Effects of Opium Addiction and Cigarette Smoking on Hematological Parameters.

BACKGROUND: The aim of the present study was to investigate the effects of opium addiction and cigarette smoking on the complete blood count (CBC). METHODS: Eighty-six male subjects, including 31 opium-addicted cigarette smokers (OACS), 19 opium-addicted non-cigarette smokers (OANCS), 17 non-opium-addicted cigarette smokers (NOACS), and 19 non-opium-addicted non-cigarette smokers (NOANCS) participated in this study. The CBC test was measured in all individuals. FINDINGS: The OACS had significantly higher white blood cell (WBC), lymphocyte, and red blood cell (RBC) count but lower in mean corpuscular volume (MCV) compared to NOANCS. The OANCS had significantly higher lymphocyte in comparison with NOACS. Our results demonstrated that the number of WBC, lymphocytes, and RBC were significantly higher, while, MCV was lower in OANCS subjects when compared to NOACS. The OACS had significantly higher level of lymphocyte in comparison with NOACS. The mean number of lymphocyte in OANCS was found significantly higher than NOACS. The smokers were shown to have significantly higher levels of WBC compared to NOANCS. CONCLUSION: Our results showed that opium-addiction, especially when associated with cigarette smoking, has intensive effects on hematological factors and these alteration might leads to greater risk for developing atherosclerosis, cardiovascular diseases, and imbalance in immune system.

Induction of apoptosis by Trichostatin A in human breast cancer cell lines: involvement of 15-Lox-1.

15-Lipoxygenase-1 (15-Lox-1) is a key enzyme mediating oxidative metabolism of polyunsaturated fatty acids and has attracted considerable interest as a potential target for the induction of apoptosis in cancer cells. Knowledge of relationship between 15-Lox-1 and histone deacetylase inhibitors is lacking in the breast cancer. This study is aimed to investigate the role of Trichostatin A (TSA) and 13(S)-HODE, as a metabolite of 15-Lox-1, in the regulation of breast cancer cell growth. The cytotoxic effect of TSA, as a potent HDAC inhibitor, was measured using MTT assay. Annexin V-FITC and PI staining were performed to detect apoptosis and cell cycle distribution using Flow cytometry. The role of 15-Lox-1 in the regulation of cell growth was assessed by 15-Lox-1 inhibitor and the level of 15-Lox-1 metabolite was measured to determine 15-Lox activity after treatment by TSA. The results demonstrated that TSA induced cell growth inhibition via 15-Lox-1, in a dose- and time-dependent manner, and subsequently accompanied by the cell cycle arrest and induction of apoptosis. Moreover, growth inhibitory effect of TSA was associated with the elevation of 15-Lox-1 metabolite (13(S)-HODE). This study provided evidences that the inhibitory effect of TSA on the breast cancer cell growth occurs via the induction of 15-Lox-1 activity and 13(S)-HODE production. Our findings underline the possible role of 15-Lox-1/13(S)-HODE pathway as a promising molecular approach for the induction of apoptosis in breast cancer cells.

Hematological changes in opium addicted diabetic rats.

BACKGROUND: Chronic opioid treatment in animal models has shown to alter hematological parameters. OBJECTIVES: The aim of this study was to evaluate the biological effects of opium on the number of peripheral blood cells and red blood cells (RBCs) indices in diabetic rats. MATERIALS AND METHODS: Peripheral blood samples were collected from diabetic, opium-addicted, diabetic opium-addicted and normal male and female rats and hematological parameters were measured. RESULTS: The mean number of white blood cells (WBCs) was significantly higher in diabetic opium-addict females compared to diabetic non-addict female group. In both male and female, the mean number of neutrophils was significantly higher and the mean number of lymphocytes was lower in diabetic opium-addicted rats than those observed in diabetic non-addicted group. In diabetic opium-addicted male group the mean counts of RBC significantly increased as compared with diabetic male group. However, in diabetic addicted female, the mean number of RBCs was significantly lower than diabetic non-addicted female group. In both males and females, the mean number of platelets was significantly lower in diabetic addict rats compared to diabetic non-addict group. CONCLUSIONS: Generally, the results indicated that opium addiction has different effects on male and female rats according to the number of WBC, RBC and RBC indices. It could also be concluded that in the opium-addicts the risk of infection is enhanced due to the weakness of immune system as a result of the imbalance effect of opium on the immune cells.

Ovary cells apoptosis in opium-addicted diabetic and non-diabetic rats.

BACKGROUND: Apoptosis is a physiological mechanism of cell death and it can be triggered by a variety of internal and external stimuli. It has been indicated that some opium derivatives develop cell apoptosis. OBJECTIVES: The aim of this investigation was to evaluate the effect of opium addiction on ovary cell apoptosis in diabetic and non-diabetic Wistar rats. MATERIALS AND METHODS: This experimental study was done on control, control-addicted, diabetic and diabetic-addicted rats. DNA fragmentation as a biomarker of apoptosis was determined by the TUNEL assay. RESULTS: The blood glucose concentration in diabetic-addicted and diabetic rats was increased when compared to control (P < 0.001). There was no significant difference between weights of control, control-addicted (non-diabetic) and diabetic-addicted groups during this study. The results of this study indicated that apoptosis in addicted and diabetic-addicted ovary cells was significantly higher than in diabetic group, and also apoptosis in addicted group was significantly more than the control rats. In addition, we found that ovary cells apoptosis of diabetic rats were significantly less than in control group. CONCLUSIONS: Overall, these findings suggest that opium-addiction could play an important role in ovary cell apoptosis and could be very harmful for the reproductive system. Also, ovary cells of non-diabetic rats are more susceptible to opium-induced apoptosis than those of diabetic.