Diosgenin potentiates the anticancer effect of doxorubicin and volasertib via regulating polo-like kinase 1 and triggering apoptosis in hepatocellular carcinoma cells.

A common approach to cancer therapy is the combination of a natural product with chemotherapy to overcome sustained cell proliferation and chemotherapy resistance obstacles. Diosgenin (DG) is a phytosteroidal saponin that is naturally present in a vast number of plants and has been shown to exert anti-cancer activities against several tumor cells. Herein, we assessed the chemo-modulatory effects of DG on volasertib (Vola) as a polo-like kinase 1 (PLK1) inhibitor and doxorubicin (DOX) in hepatocellular carcinoma (HCC) cell lines. DOX and Vola were applied to two human HCC cell lines (HepG2 and Huh-7) alone or in combination with DG. The cell viability was determined, and gene expressions of PLK1, PCNA, P53, caspase-3, and PARP1 were evaluated by RT-qPCR. Moreover, apoptosis induction was determined by measuring active caspase-3 level using ELISA method. DG enhanced the anticancer effects of Vola and DOX. Moreover, DG enhanced Vola- and DOX-induced cell death by downregulating the expressions of PLK1 and PCNA, elevating the expressions of P53 and active caspase-3. DG showed promising chemo-modulatory effects to Vola and DOX against HCC that may be attributed partly to the downregulation of PLK1 and PCNA, upregulation of tumor suppressor protein P53, and apoptosis induction. Thus, DG combination with chemotherapy may be a promising treatment approach for HCC.

Promising renoprotective effect of gold nanoparticles and dapagliflozin in diabetic nephropathy via targeting miR-192 and miR-21.

Diabetic nephropathy (DN) is a worldwide issue that eventually leads to end-stage renal failure, with limited therapeutic options. Prior research has revealed that gold nanoparticles (AuNPs) have a substantial antidiabetic impact. In addition, sodium-glucose cotransporter2 (SGLT2) inhibitors, including dapagliflozin (DAPA), had renoprotective impact on DN. Therefore, this research attempted to determine the potential AuNPs and DAPA impacts in ameliorating experimentally DN induction and the underlying mechanisms focusing on miR-192 and miR-21, correlating them with autophagy, apoptosis, fibrosis, and oxidative stress. Diabetes induction was through a single intraperitoneal streptozotocin (55 mg/kg) injection, and rats with diabetes received AuNPs (2.5 mg/kg/day) as well as DAPA (2 mg/kg/day) for 7 weeks as a treatment. AuNPs and DAPA treatment for 7 weeks substantially alleviated DN. AuNPs and DAPA significantly increased catalase (CAT) activity as well as serum total antioxidant capacity (TAC), along with a substantial decline in malondialdehyde (MDA). AuNPs and DAPA treatment alleviated renal fibrosis as they decreased transforming growth factorß1(TGF-ß1) as well as matrix metalloproteinase-2 (MMP-2) renal expression, decreased apoptosis through alleviating the proapoptotic gene (caspase-3) renal expression and increased the antiapoptotic gene (Bcl-2) renal expression, and increased autophagy as they increased LC-3 as well as Beclin-1 renal expression. Autophagy activation, inhibition of apoptosis, and renal fibrosis could be due to their inhibitory impact on miR-192 and miR-21 renal expression. AuNPs and DAPA have a protective effect on DN in rats by targeting miR-192 and miR-21 and their downstream pathways, including fibrosis, apoptosis, autophagy, and oxidative stress.

Sinapic acid and 3,3'-diindolylmethane potentiate cyclophosphamide antitumor activity through induction of apoptosis and inhibition of metastasis.

AIM: New therapeutic strategies are required to enhance the anticancer efficacy of chemotherapeutic drugs and to reduce their cytotoxicity. The purpose of this study was to assess the anti-tumor, antimetastatic and anti-apoptotic activities of sinapic acid (SA) and 3,3'-diindolylmethane (DIM) in solid Ehrlich carcinoma (SEC) induced in mice and combining SA or DIM compounds with cyclophosphamide (CYP). METHODS: For induction of solid tumor, the right hind limbs of mice were inoculated subcutaneously with Ehrlich carcinoma cells. After 5 days of tumor inoculation, mice were treated with SA (56 mg/kg), DIM (40 mg/kg), CYP (10 mg/kg), and their combinations (SA/CYP) and (SA/DIM) for 21 days. The mRNA levels of Elabela, Serpina3, caspase-3, MMP-2 and MMP-9 were assessed by qPCR. Tumor and liver tissues were stained with hematoxylin and eosin for histological examination. Serum was investigated for ALT and AST activities. MAIN FINDINGS: Treatment of SEC mice with SA and DIM significantly reduced solid tumor weight by 45.6% and 33.2%, respectively. They also reduced tumor size and increased life span of SEC mice. SA and DIM diminished area of metastatic nodules of tumor cells in the liver by 54.1% and 47.4%, respectively. They also reduced serum aminotransferases activities. Both SA and DIM were found to upregulate caspase 3 and downregulate MMP-2 and MMP-9. Furthermore, SA and DIM reduced gene expression of Elabela by (44.8% and 35.1%) and Serpina3 by (30.7% and 23.5%), respectively. SA and DIM were also shown to potentiate the anti-tumor activity CYP. SA and DIM showed promising antitumor effects and enhanced CYP antitumor activity mostly through upregulation of apoptotic caspase 3 and suppressing metastatic enzymes MMP-2 and MMP-9. Additionally, SA and DIM exhibited a hepatoprotective effect. Our results suggest that these natural compounds may be used to improve the efficacy and reduce the adverse effects of chemotherapeutic drugs in the treatment of solid malignancies.

Promising cardioprotective effect of baicalin in doxorubicin-induced cardiotoxicity through targeting toll-like receptor 4/nuclear factor-κB and Wnt/ß-catenin pathways.

Doxorubicin (Dox) is an indispensable chemotherapeutic agent associated with damaging cardiotoxicity. Baicalin (BA) is a flavonoid, extracted from the medicinal plant Scutellariae baicalensis Georgi. BA is well known for its anti-inflammatory and antioxidant effects. Our study investigated the potential effect of BA in attenuating Dox-induced cardiotoxicity. To this end, male Swiss albino mice were given BA (100 mg/kg/d, orally) for 4 wk and were challenged with Dox (six intraperitoneal doses, each 2.5 mg/kg, every other day with a final cumulative dose of 15 mg/kg). Serum activities of cardiac biomarkers (cardiac troponin-I, creatine kinase-membrane bound, lactate dehydrogenase, and aspartate aminotransferase) were assessed along with the histopathological examination of the heart tissues. Gene expression of Toll-like receptor 4 (TLR4) was analyzed by quantitative reverse transcription real-time polymerase chain reaction. Analysis of the protein levels of ß-catenin and nuclear factor-κB (NF-κB) was done immunohistochemically. Cardiac Dickkopf-1 (DKK1) and interleukin-1beta (IL-1ß) were quantified by enzyme-linked immuno-sorbent assay. Cardiac levels of reduced glutathione (GSH) and malondialdehyde (MDA) were detected spectrophotometrically. Pretreatment with BA significantly prevented Dox-induced elevation of serum activities of cardiac biomarkers and alterations to the heart. Moreover, BA suppressed the gene overexpression of cardiac TLR4 and subsequently prevented Dox-induced elevation of both cardiac NF-κB and IL-1ß. BA also significantly reduced the cardiac levels of DKK1 and elevated the level of ß-catenin. Dox-induced elevation of MDA and reduction of GSH were reversed by BA. BA exhibited a novel cardioprotective effect against Dox-induced cardiotoxicity. The cardioprotective effect was indicated through the inhibition of the inflammatory TLR4/NF-κB pathway and the activation of the protective Wnt/ß-catenin pathway by the suppression of DKK1.

A prospective trial investigating the role of Serum 25-Hydroxyvitamin D in diagnosis and prognosis of bladder cancer.

PURPOSE: Higher levels of serum 25-hydroxyvitamin D 25(OHD) are associated with better prognosis in breast and colorectal cancer. However, the evidence is still inconclusive for bladder cancer (BC). Herein, we investigated the diagnosis and prognosis roles of serum levels of 25(OHD) in suspected BC patients presented by hematuria. METHODS: This prospective cohort study involved suspected patients of BC presented with hematuria. Patients were evaluated by CT urogram, office cystoscopy and urine cytology with subsequent inpatient biopsy for positive findings. Baseline blood samples were collected for measurement of 25(OHD) by electrochemiluminescence binding assay at the time of diagnosis. Patients with non-muscle-invasive BC (NMIBC) underwent transurethral resection of bladder tumor (TURBT) and adjuvant intravesical chemotherapy or BCG instillation. Patients were followed up for their recurrence status during 10 to 24 months. Recurrence was defined as the first time of NMIBC pathological relapse during the follow up period. RESULTS: A total of 115 patients were included in the final analysis. Patients had proven pathological BC (64 with NMIBC, and 20 with muscle invasive) and 31 patients were considered as control group. Controls were those patients with BC-free workup (including cytology, cystoscopy, and upper tract imaging). BC group showed a lower level of 25(OHD) than control group 16.47±5.88 versus 28.99±3.19 ng/mL (p<0.001). In addition, muscle invasive group also showed a lower level than NMIBC group 13.17±4.5 versus 17.49±5.04 ng/mL (P = 0.003). During the follow-up period of, tumor recurrence occurred in 16 (25%) of NMIBC patients. The baseline 25(OHD) were decreased in patients who experienced early recurrence; without being statistically significant (15.99 ± 5.17 vs. 18.38 ± 5.14 ng/mL; p = 0.08). 25(OHD) deficiency/insufficiency occurred in 5 (16.1%) and 64 (76.2%) in control and BC patients, respectively, (odds-ratios (OR): 2.13; 95% confidence intervals (CI), 1.52-2.99; P < 0.0001). CONCLUSION: Serum 25(OHD) is significantly decreased in BC patients especially those with tumor muscle invasive group. However, the baseline serum 25(OHD) does not predict the recurrence in the NMIBC patients.

Sesamol Upregulates Death Receptors and Acts as a Chemosensitizer in Solid Ehrlich Carcinoma Model in Mice.

AIMS: The aim of the present study was to investigate the anti-tumor effect of sesamol (SML), a nutritional phenolic compound of sesame, in solid Ehrlich carcinoma (SEC) model in mice and its ability to enhance doxorubicin (DOX) anti-tumor activity. Moreover, we analyzed the ability of SML to protect against DOX-induced cardiotoxicity. MAIN METHODS: SML (70 mg/kg), DOX (2 mg/kg) and their combination were given to mice bearing SEC for 21 day. The mRNA level of Fas, FasL, TRAILR2, TRAIL, caspase-3 and Bcl-2 were assessed by qPCR. Tumor and cardiac tissues were examined for histopathological changes by hematoxylin and eosin. Active caspase-3 was scored by immunohistochemical analysis. KEY FINDINGS: SML treatment significantly decreased solid tumor size and weight. In addition, SML enhanced DOX anti-tumor activity. SML treatment either alone or in combination with DOX induced upregulation of Fas/FasL and TRAILR2/TRAIL gene expression. Moreover, SML increased caspase-3 protein and gene expressions and decreased Bcl-2 gene expression. SIGNIFICANCE: SML upregulates death receptors expression and enhances apoptosis induction in tumor cells that may explain its anti-tumor activity. Not only that, but SML also enhances DOX anti-tumor activity and attenuates its cardiotoxicity.

Niclosamide-loaded polymeric micelles ameliorate hepatocellular carcinoma in vivo through targeting Wnt and Notch pathways.

AIM: Niclosamide (NIC) is an anthelmintic agent repurposed as a potent anticancer agent. However, its use is hindered by its poor solubility. We investigated the underlying mechanisms of NIC anticancer activity employing a novel oral NIC pluronic-based nanoformulation and tested its effect in thioacetamide-induced hepatocellular carcinoma (HCC) in rats. We evaluated its antitumor effect through regulating Wnt/ß-catenin and Notch signaling pathways and apoptosis. MAIN METHODS: Niclosamide-loaded pluronic nanoparticles (NIC-NPs) were optimally developed and characterized with sustained release properties up to 7 days. Sixteen weeks after HCC induction, NIC (70 mg/kg) and an equivalent dose of NIC-NPs were administered orally for 3 consecutive weeks. Hepatocyte integrity was assessed by measuring serum levels of aminotransferases, ALP, GGT, bilirubin, albumin and total protein. HCC development was detected by measuring AFP expression. Necroinflammation and fibrosis were scored by histopathological examination. Wnt/ß-catenin and Notch signaling were evaluated by measuring hepatic mRNA levels of Wnt3A, Lrp5 and Lrp6 Co-receptors, Dvl-2, Notch1 and Hes1 and ß-catenin protein levels. Apoptosis was assessed by measuring mRNA and protein levels of cyclin D1 and caspase-3. KEY FINDING: The novel NIC-NPs restored liver integrity, reduced AFP levels and showed improved anticancer and proapoptotic activities compared to drug alone. The inhibitory effect of NIC on Wnt/ß-catenin and Notch signaling pathways was potentiated by the NIC-NPs formulation. SIGNIFICANCE: We conclude that NIC acts by inhibiting Wnt/ß-catenin and Notch signaling and inducing apoptosis in HCC. Developing pluronic-based nanoformulations may be a promising approach to improve NIC solubility and offer the possibility of controlled release.

Thymoquinone potentiates miR-16 and miR-375 expressions in hepatocellular carcinoma.

AIMS: MicroRNAs (miRNAs) are non-coding RNAs that control post-transcriptional gene expression. Recently, miRNAs were confirmed to be promising biomarkers for different pathological conditions. This study assessed the role of serum miR-16 and miR-375 in HCC development in chronic liver disease patients such as cirrhosis. Moreover, miR-16 and miR-375 levels were estimated in HCC cell lines (HepG2 and Huh7) after treatment with doxorubicin (DOX), thymoquinone (TQ) and their combination. MAIN METHODS: Serum miR-16 and miR-375 were analyzed in 30 HCC patients, 20 cirrhosis patients and 10 healthy volunteers using RT-PCR. Moreover, HepG2 and Huh7 cells were incubated with DOX, TQ or TQ/DOX combination for 24 h and the levels of miR-16, miR-375 and gene expression of anti-apoptotic protein BCL-2 were determined in cell lysates using RT- PCR. Moreover, the ability of DOX, TQ and TQ/DOX combination to induce apoptosis were analyzed by measuring caspase-3 expression using ELISA method. KEY FINDINGS: Serum miR-16 and miR-375 levels were significantly decreased in HCC patients as compared to cirrhosis and healthy control group. Also, combined use of serum miR-16 and miR-375 showed a better predictive ability than each alone. Moreover, the expression level of miR-16 and miR-375 in HepG2 and Huh7 cells increased significantly after treatment with DOX and TQ. Also, TQ/DOX combination improved apoptosis by increasing caspase-3 expression and decreasing of BCL-2 expression. SIGNIFICANCE: This study proved that the combined use of serum miR-16 and miR-375 was better than each alone for HCC detection. Moreover, TQ induced apoptosis and upregulatedmiR-16 and miR-375 expression in HCC cells that may explain its anticancer activity.

Polo-like kinase 1 as a promising diagnostic biomarker and potential therapeutic target for hepatocellular carcinoma.

Hepatocellular carcinoma is a major cause of cancer mortality worldwide. The outcome of hepatocellular carcinoma depends mainly on its early diagnosis. To date, the performance of traditional biomarkers is unsatisfactory. Polo-like kinase 1 is a serine/threonine kinase that plays essential roles in cell cycle progression and deoxyribonucleic acid damage. Moreover, polo-like kinase 1 knockdown decreases the survival of hepatocellular carcinoma cells; therefore, polo-like kinase 1 is an attractive target for anticancer treatments. Nobiletin, a natural polymethoxy flavonoid, exhibits a potential antiproliferative effect against a wide variety of cancers. This study targets to identify a reliable diagnostic biomarker for hepatocellular carcinoma and provide a potential therapeutic target for its treatment. Polo-like kinase 1 levels were analyzed in 44 hepatocellular carcinoma patients, 33 non-hepatocellular carcinoma liver cirrhosis patients and 15 healthy controls using the enzyme-linked immunosorbent assay method. Receiver operating characteristics curve analysis was used to establish a predictive model for polo-like kinase 1 relative to α-fetoprotein in hepatocellular carcinoma diagnosis. Furthermore, in the in vitro study, gene expressions were assessed by quantitative polymerase chain reaction in two human hepatocellular carcinoma cell lines after treatment with doxorubicin and polo-like kinase 1 inhibitor volasertib (Vola) either alone or in combination with nobiletin. Cell viability was also determined using the crystal violet assay.: Serum polo-like kinase 1 levels in hepatocellular carcinoma patients were significantly higher than liver cirrhosis and control groups (p < 0.0001). Polo-like kinase 1 showed a reasonable sensitivity, specificity, positive predictive value, and negative predictive value in hepatocellular carcinoma diagnosis. Moreover, nobiletin improved inhibition of cell growth induced by Vola and doxorubicin. Regarding reverse transcription polymerase chain reaction results, nobiletin suppressed expressions of polo-like kinase 1 and proliferating cell nuclear antigen and elevated expressions of P53, poly (ADPribose) polymerase 1, and caspase-3. Nobiletin/doxorubicin and nobiletin/Vola showed a significant increase in caspase-3 activity indicating cell apoptosis. Polo-like kinase 1 may be a potential biomarker for hepatocellular carcinoma diagnosis and follow-up during treatment with chemotherapies. In addition, nobiletin synergistically potentiates the doxorubicin and Vola-mediated anticancer effect that may be attributed partly to suppression of polo-like kinase 1 and proliferating cell nuclear antigen expression and enhancement of chemotherapy-induced apoptosis.

Dickkopf-1 and Amphiregulin as Novel Biomarkers and Potential Therapeutic Targets in Hepatocellular Carcinoma.

Background: Hepatocellular carcinoma (HCC) is a highly fatal tumor which represents a major health problem worldwide. Due to asymptomatic nature of HCC, most patients present with the progressive stage of disease, so, unfortunately, there are no effective therapies. Existing techniques for HCC surveillance and diagnosis lack the required accuracy. Therefore, searching for new diagnostic and/or therapeutic tools could improve patient survival. This study aimed to estimate the diagnostic role of Dickkopf-1 (DKK1) and amphiregulin (AREG) and to find out their correlation with different clinicopathological parameters in HCC patients. Materials and Methods: Serum levels of DKK1 and AREG in 55 HCC patients, 20 cirrhotic patients, and 15 healthy subjects as control group were measured using the ELISA technique. Results: Both of DKK1 and AREG showed a significant increase in the HCC group compared to cirrhotic and healthy groups. DKK1 at a cutoff point of 8.92 ng/ml showed that the area under the curve (AUC) was 0.826 with 87.3% sensitivity and 82.9% specificity. DKK1 showed a significant correlation with tumor size, liver dysfunction, and poor performance status in HCC patients. AREG at a cutoff point of 8.74 pg/ml showed a sensitivity of 74.5% but low specificity (47.1%). AREG showed a significant correlation with portal vein thrombosis and tumor metastasis in HCC patients. Conclusion: Serum DKK1 could be a diagnostic biomarker for HCC. Both of DKK1 and AREG may play significant roles in tumor progression and may offer promising therapeutic targets in HCC patients.

P-Coumaric acid alleviates experimental diabetic nephropathy through modulation of Toll like receptor-4 in rats.

AIMS: Diabetic nephropathy (DN) is responsible for the occurrence of 30-47% of the incident cases of end-stage renal disease (ESRD) worldwide. DN is a chronic inflammatory disorder, which results from hyperglycemia-induced alterations and leads to renal fibrosis and ESRD. Toll like receptor-4 (TLR-4) participates in regulation of inflammatory response through controlling of innate immune system. P-Coumaric Acid (P-CA) is a natural hydroxycinnamic acid derivative and is widely present in vegetables, fruits, mushrooms and cereals. This study aimed to explore the renoprotective effect of P-CA, as anti-inflammatory and antioxidant natural compound, against experimental DN. METHODS: DN was induced by single intraperitoneal injection of streptozotocin (45 mg/kg) in rats. In kidney homogenate, levels of TLR-4, interleukin-6 (IL-6) and transforming growth factor ß1 (TGFß1) were measured using ELISA technique. Also, kidney collagen content was determined colorimetrically. KEY FINDINGS: Oral administration of P-CA (100 mg/kg) for 8 weeks significantly alleviated the DN. P-CA significantly reduced serum concentrations of glucose, creatinine, blood urea nitrogen (BUN) and reduced protein content in urine. Also, P-CA significantly increased superoxide dismutase (SOD) activity and significantly reduced kidney contents of malondialdehyde (MDA), TLR-4, IL-6, TGFß1 and collagen when compared with DN group. Moreover, P-CA significantly improved DN-induced histopathological abnormalities. SIGNIFICANCE: P-CA confers protection against the progression of DN. This renoprotective effect can be attributed to its ability to decrease the generation of inflammatory and fibrotic cytokines in addition to restoring oxidant/antioxidant balance through its ability to down-regulate TLR-4 activation.

Thymoquinone upregulates TRAIL/TRAILR2 expression and attenuates hepatocellular carcinoma in vivo model.

AIMS: Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related mortality worldwide. Indeed, chemotherapeutic drugs-induced systemic toxicity results in suboptimal cancer treatment. Consequently, there is a need for exploring of a safe and effective therapy for cancer patients. This study aimed to evaluate the hepatoprotective effect of thymoquinone (TQ) against thioacetamide (TAA)-induced HCC. Also, we investigated TQ's ability to sensitize cancer cells toward TRAIL/TRAILR2 apoptotic pathway. MAIN METHODS: Forty male Sprague Dawley rats were divided into 4 groups (n = 10) as follows: control group, CMC group, HCC group and HCC + TQ group. Serum levels of liver function biomarkers and Alpha-Fetoprotein (AFP), as well as hepatic levels of glutathione (GSH) and Alpha-Fetoprotein (MDA) were measured. Transforming growth factor-beta 1 (TGF-ß1), TRAILR2, TRAIL, caspase-3, caspase-9, caspase-8 and B cell lymphoma-2 (Bcl-2) mRNA levels were assessed by Quantitative, Real-Time PCR. Fibrosis percentage and necroinflammation were quantified by histopathological examination. KEY FINDINGS: Our results indicated improvement in liver functions, decrease in AFP level and attenuation of HCC progression in TQ treated rats. TQ upregulated TRAIL/TRAILR2 and subsequently enhanced apoptosis as hinted by caspase-3 upregulation and Bcl-2 downregulation. Also, TQ decreased TGF-ß1 gene expression level. Moreover, HCC + TQ group showed significant increase in hepatic GSH level and marked decrease in hepatic MDA level. SIGNIFICANCE: This study proved that TQ is able to suppress HCC development via decreasing oxidative stress, suppression of TGF-ß1 and induction of TRAIL-mediated apoptosis.

The co-chaperone UNC45A is essential for the expression of mitotic kinase NEK7 and tumorigenesis.

Cumulative evidence suggests that the heat shock protein 90 (Hsp90) co-chaperone UNC-45 myosin chaperone A (UNC45A) contributes to tumorigenesis and that its expression in cancer cells correlates with proliferation and metastasis of solid tumors. However, the molecular mechanism by which UNC45A regulates cancer cell proliferation remains largely unknown. Here, using siRNA-mediated gene silencing and various human cells, we report that UNC45A is essential for breast cancer cell growth, but is dispensable for normal cell proliferation. Immunofluorescence microscopy, along with gene microarray and RT-quantitative PCR analyses, revealed that UNC45A localizes to the cancer cell nucleus, where it up-regulates the transcriptional activity of the glucocorticoid receptor and thereby promotes expression of the mitotic kinase NIMA-related kinase 7 (NEK7). We observed that UNC45A-deficient cancer cells exhibit extensive pericentrosomal material disorganization, as well as defects in centrosomal separation and mitotic chromosome alignment. Consequently, these cells stalled in metaphase and cytokinesis and ultimately underwent mitotic catastrophe, phenotypes that were rescued by heterologous NEK7 expression. Our results identify a key role for the co-chaperone UNC45A in cell proliferation and provide insight into the regulatory mechanism. We propose that UNC45A represents a promising new therapeutic target to inhibit cancer cell growth in solid tumor types.

Phenethyl isothiocyanate Triggers Apoptosis, Combats Oxidative Stress and Inhibits Growth of Ehrlich Ascites Carcinoma Mouse Model.

The aim of this study is to investigate the antitumor activity and possible molecular mechanism of Phenethyl isothiocyanate (PEITC) against Ehrlich ascites carcinoma in-vivo and in-vitro. In-vivo, ascetic fluid volume, body weight, serum malondialdehyde (MDA) level and total antioxidant capacity (TAC) were determined using Ehrlich ascites carcinoma (EAC) bearing mice. In-vitro, MTT assay was used. RT-PCR was used to investigate role of PEITC in apoptosis by analyzing the expression of Bax, caspase-9, and Bcl-2 genes. The effect of PEITC on caspase-9 enzyme activity was also tested. PEITC and/or Doxorubicin (Dox) treatment significantly suppressed EAC growth as compared to EAC/oil control mice. PEITC treatment showed a dose-dependent inhibition of EAC cells as indicated by MTT assay. We found that significant increase in MDA level and decrease in TAC caused by Dox treatment were significantly reduced by combination with PEITC treatment. Bax, caspase-9 genes' expression and caspase-9 enzymatic activity were significantly increased, while Bcl-2 gene expression was significantly decreased in PEITC treated mice. PEITC may act as a promising anticancer agent either alone or more effectively in combination with Dox through apoptotic cell death induction.

Vitamin D potentiates anti-tumor activity of 5-fluorouracil via modulating caspase-3 and TGF-ß1 expression in hepatocellular carcinoma-induced in rats.

We investigated the role of vitamin D (Vit D) alone and in combination with 5-fluorouracil (5-FU) in thioacetamide (TAA)-induced hepatocellular carcinoma (HCC) in rats. Fifty male Sprague-Dawley rats were randomized into a control group and 4 groups that received TAA (200 mg/kg, i.p.) twice per week for 16 weeks. These 4 groups were further divided as follows: HCC group; 5-FU group (75 mg/kg, i.p., once weekly for 3 weeks starting from the 12th week); Vit D group (200 IU/kg daily by oral tube for 16 weeks); and 5-FU + Vit D group (received the previously mentioned dosage regimens of 5-FU and Vit D). HCC was detected by histopathological changes in liver sections and the elevation of serum α-fetoprotein (AFP). Treatment with 5-FU + Vit D significantly decreased gene expression of nuclear factor erythroid 2-related factor 2 (NrF2) and transforming growth factor ß1 (TGF-ß1) at both the gene and protein level and serum AFP concentrations in comparison with their corresponding monotherapy. Moreover, 5-FU + Vit D treatment enhanced apoptosis by increasing caspase-3 gene and protein expression. Conclusively, Vit D enhances antitumor activity of 5-FU in an HCC-induced model and improves liver function of treated animals. Combination therapy in a TAA-induced HCC rat model was more effective than 5-FU or Vit D through the modulation of TGF-ß1, caspase-3, and NrF2 expressions.

Diagnostic and Prognostic Value of Talin-1 and Midkine as Tumor Markers in Hepatocellular Carcinoma in Egyptian Patients

Background: Hepatocellular carcinoma (HCC) is a main cause of cancer death all over the world. Treatment and outcome of HCC based on its early diagnosis. This study was conducted to estimate the role of talin-1 and midkine in combination with total antioxidant capacity (TAC) as tumor markers in HCC patients. Methods: Serum levels of talin-1 and midkine were measured in 90 Egyptian subjects including 44 patients with HCC, 31patients with cirrhosis and 15 healthy controls using enzyme-linked immunosorbent assay (ELISA) technique. While a colorimetric method was used for measurement of TAC. Results: Serum talin-1 in HCC patients was significantly lower than that in patients with cirrhosis (P<0.001) and normal control (P<0.001). In addition, increased invasion and metastasis correlated with reduced talin-1 level. Serum midkine in HCC patients was significantly higher compared to cirrhotic patients (P<0.001) and normal control (P<0.001). Midkine at a cut off value of 1683 pg/ml showed a sensitivity of (81.82%) and specificity of (83.87%). While alpha-fetoprotein (AFP) at a cut off value of 200 ng/ml had a sensitivity of (52.27%), while specificity was (96.77%). Midkine was positive in 80.9% of HCC patients with negative AFP. Serum TAC was significantly decreased in HCC patients when compared with control group (P<0.001). Conclusion: Talin-1 may be implicated in the carcinogenesis and metastasis of HCC and can be used as a useful tumor marker for HCC. Midkine may be a potential diagnostic marker for HCC and may be used in addition to AFP to increase the sensitivity of HCC detection.

Silymarin and caffeine combination ameliorates experimentally-induced hepatic fibrosis through down-regulation of LPAR1 expression.

AIMS: Lysophosphatidic acid is a lipid mediator that is supposed to be implicated in hepatic fibrosis. Silymarin and caffeine are natural compounds known for their anti-inflammatory and antioxidant effects. Our study aimed to explore the effect of silymarin, caffeine, and their combination on lysophosphatidic acid receptor 1 (LPAR1) pathway in thioacetamide (TAA)-induced hepatic fibrosis. MAIN METHODS: Hepatic fibrosis was induced in male Sprague-Dawley rats by intraperitoneal injection of 200 mg/kg of TAA twice a week for 8 weeks. Silymarin (50 mg/kg), caffeine (50 mg/kg), and their combination (50 mg/kg silymarin + 50 mg/kg caffeine) were orally given to rats every day for 8 weeks along with TAA injection. Liver functions were measured. Histopathological examination of liver tissues was performed using hematoxylin and eosin and Masson's trichrome staining. mRNA expressions of LPAR1, transforming growth factor beta 1 (TGF-ß1), connective tissue growth factor (CTGF), and alpha smooth muscle actin (α-SMA) were measured using RT-PCR. LPAR1 tissue expression was scored using immunohistochemistry. KEY FINDINGS: Silymarin, caffeine, and their combination significantly improved liver function. They caused significant decrease in fibrosis and necro-inflammatory scores. Combination of silymain and caffeine caused a significant decrease in the necro-inflammatory score than the single treatment with silymarin or caffeine. In addition, silymarin, caffeine, and their combination significantly decreased hepatic LPAR1, TGF-ß1, CTGF, and α-SMA gene expressions and LPAR1 tissue expression. SIGNIFICANCE: Silymarin, caffeine, and their combination protect against liver fibrosis through down-regulation of LPAR1, TGF-ß1, and CTGF.

Modulating effects of omega-3 fatty acids and pioglitazone combination on insulin resistance through toll-like receptor 4 in type 2 diabetes mellitus.

Toll-like receptor 4 (TLR-4) plays important roles in innate immunity. Changes in the reduction-oxidation balance of tissues can lead to a pro-inflammatory state and insulin resistance. An action thought to be mediated by TLRs. Omega-3 fatty acids and Peroxisome Proliferator Activated Receptor gamma (PPAR-γ) agonists as pioglitazone are used for decreasing inflammation. The aim of this study is to investigate the anti-diabetic effects of combining omega -3 fatty acid with pioglitazone on type 2 diabetes, and the modifying effects on TLR-4. Type 2 diabetes was induced in male Sprague-Dawley rats by combination of high fat diet and low dose streptozotocin (35mg/kg). Diabetic rats were treated with omega-3 fatty acids (10% W/W diet), pioglitazone (20mg/kg), and their combination for 4 weeks. Omega-3 fatty acids and the combination treatment significantly decreased TLR-4 activation. Omega-3 fatty acids, pioglitazone, and their combination significantly decreased TLR-4 mRNA expression, hepatic malondialdehyde, total cholesterol and triglycerides levels, compared to diabetic group. Pioglitazone and the combination significantly decreased blood glucose levels and improved insulin resistance. In conclusion, combining omega-3 fatty acids with pioglitazone showed potential effects in lowering blood glucose levels and improving lipid profile and insulin resistance. Such effects are mediated through modulation of TLR-4.

Chloroquine upregulates TRAIL/TRAILR2 expression and potentiates doxorubicin anti-tumor activity in thioacetamide-induced hepatocellular carcinoma model.

Impaired apoptosis and systemic toxicity of chemotherapeutic drugs make cancer treatment suboptimal. Thus, there is urgency for drug repurposing which facilitates discovery of safe and effective combination therapy. This study aimed to evaluate chloroquine's (CQ) ability to trigger TRAIL/TRAILR2 apoptotic pathway in thioacetamide (TAA)-induced hepatocellular carcinoma (HCC) either alone or in combination with doxorubicin (DOX). Moreover, its ability to attenuate DOX-induced cardiotoxicity was investigated. TAA was injected in male Sprague Dawely rats (200 mg/kg; ip; 2 times/week) for 16 weeks. After the 16th week, rats were further divided into different groups (n = 10) and treated for 7 weeks. CQ group (received CQ 25 mg/kg/day; orally), DOX group (received DOX 1 mg/kg; ip; 2 times/week) and CQ/DOX group. Liver function biomarkers, AFP, hepatic levels of MDA and GSH, serum CK-MB and LDH enzymes activity were measured. Quantitative, Real-Time PCR was used to measure TRAIL, TRAILR2, caspase-8, caspase-9, caspase-3, BCL-2 and TGF-ß1 genes expression levels. Necroinflammation and fibrosis were scored by histopathological examination. CQ improved liver functions, reduced AFP level and attenuated HCC progression. CQ induced apoptosis via upregulation of TRAIL/TRAILR2, caspase-8, caspase-3 and caspase-8 genes and downregulation of BCL-2 gene. Moreover, CQ/DOX showed marked decrease in hepatic MDA level, serum CK-MB, LDH enzymes activity, as well as marked increase in hepatic GSH level. In conclusion, this work assess the in vivo efficacy of CQ/DOX combination therapy in this HCC model that not only has enhanced anti-tumor activity but it also protects against DOX-induced cardiotoxicity. Nevertheless, more studies should be performed to illustrate the molecular mechanism of CQ's cardioprotective effect.