Anticancer Effect of Theranekron® on Androgen-Dependent Prostate Cancer Cells.

Objectives: Prostate cancer (PCa) is a significant health problem in men worldwide. Although there are numerous treatment choices for PCa, acquired resistance limits treatment success. Therefore, there is a need for new approaches as powerful resources for use in alternative or supportive therapeutic strategies for anticancer therapeutics. Theranekron® is a commercially available alcoholic extract of Tarantula cubensis. Recent studies have shown the potent anticancer effect of theranekron in human tumors, including PCa. Herein, we comparatively examined the antiproliferative activity of theranekron and its biochemical action on androgenic signaling and cell cycle-related cyclin proteins in androgen-dependent PCa cells, LNCaP, VCaP, and 22Rv1. Materials and Methods: Human androgen-dependent PCa cells, LNCaP (CRL-1740TM), 22Rv1 (CRL-2505TM), and VCaP (CRL-2876TM) were used to evaluate the effect of theranekron in vitro. The impact of theranekron on cell viability was evaluated using a WST-1-based viability test. Its impact on AR, cyclin A2, cyclin B1, and cyclin E1 was examined by immunoblotting. To test the anti-malignant effect of theranekron on 3D tumor formation of PCa cells, soft agar assay was used. Results: Our results indicated that theranekron treatment significantly reduced the viability of PCa cells. It remarkably decreased the protein levels of AR, cyclin A2, cyclin B1, and cyclin E1 in a dose-dependent manner. In addition, Theranekron administration strongly limited the 3D tumor formation of LNCaP, 22Rv1, and VCaP cells. Conclusion: Our findings strongly suggest that theranekron may offer potent therapeutic efficacy against androgen-dependent PCa cells. Moreover, it may be a potent component for preventing acquired resistance to chemotherapeutics.

New mode of action of curcumin on prostate cancer cells: Modulation of endoplasmic reticulum-associated degradation mechanism and estrogenic signaling.

Prostate cancer is leading to cancer-related mortality in numerous men each year worldwide. While there are several treatment options, acquired drug resistance mostly limits the success of treatments. Therefore, there is a need for the development of innovative treatments. Curcumin is one of the bioactive polyphenolic ingredients identified in turmeric and has numerous biological activities, such as anti-inflammatory and anticancer. In the present study, we investigated the effect of curcumin on the ER-associated degradation (ERAD) and estrogenic signaling in prostate cancer cells. The antiproliferative effect of curcumin on human androgen-dependent prostate cancer cell lines LNCaP and VCaP was estimated by WST-1 assay. Morphological alterations were investigated with an inverted microscope. We investigated the effect of curcumin on ERAD and estrogen signaling proteins by immunoblotting assay. To evaluate the impact of curcumin on endoplasmic reticulum (ER) protein quality-related, the expression level of 32 genes was analyzed by quantitative reverse transcription polymerase chain reaction. The nuclear translocation of estrogen receptor was examined by nuclear fractionation and immunofluorescence microscopy. We found that curcumin effectively reduced the proliferation rates of LNCaP and VCaP cells. ERAD proteins; Hrd1, gp78, p97/VCP, Ufd1 and Npl4 were strongly induced by curcumin. Also, the steady-state level of polyubiquitin was increased in a dose-dependent manner in both cell lines. Curcumin administration remarkably decreased the protein levels of estrogen receptor-alfa (Erα), whereas estrogen receptor-beta unaffected. Additionally, curcumin strongly restricted the nuclear translocation of Erα. Present data suggest that curcumin may be effectively used in therapeutic approaches associated with the targeting ER protein quality control mechanism and modulation of estrogen signaling in prostate cancer.

HERPUD1, a Member of the Endoplasmic Reticulum Protein Quality Control Mechanism, may be a Good Target for Suppressing Tumorigenesis in Breast Cancer Cells.

Objectives: Breast cancer is the most frequently diagnosed cancer type and the second leading cause of cancer-related death in women. Recent studies have highlighted the importance of the endoplasmic reticulum (ER) protein quality control mechanism for the survival of many cancers. It has also been recommended as a good target for the treatment of many cancer types. Homocysteine inducible ER protein with ubiquitin-like domain 1 (HERPUD1) functions as one of the main components of ER-associated degradation, which is an ER-resident protein quality mechanism. Today, the association of HERPUD1 with breast carcinogenesis is still not fully understood. Herein, we evaluated the possibility of HERPUD1 as a potential therapeutic target for breast cancer. Materials and Methods: The effects of HERPUD1 silencing on epithelial-mesenchymal transition (EMT), angiogenesis, and cell cycle proteins were analyzed by immunoblotting studies. To test the role of HERPUD1 on tumorigenic features, WST-1-based cell proliferation assay, wound-healing assay, 2D colony formation assay, and Boyden-Chamber invasion assay were performed in human breast cancer cell line MCF-7. The statistical significance of the differences between the groups was determined by Student's t-test. Results: Our results displayed that suppressing HERPUD1 expression reduced the cell cycle-related protein levels, including cyclin A2, cyclin B1, and cyclin E1 in MCF-7 cells. Also, silencing of HERPUD1 remarkably decreased expression levels of EMT-related N-cadherin and angiogenesis marker vascular endothelial growth factor A. Moreover, we determined that cell proliferation, migration, invasion, and colony formation of MCF-7 cells were significantly limited by silencing of HERPUD1. Conclusion: Present data suggest that HERPUD1 may be an effective target for biotechnological and pharmacological strategies to be developed to treat breast cancer.

Dexpanthenol protects against lipopolysaccharide-induced acute kidney injury by restoring aquaporin-2 levels via regulation of the silent information regulator 1 signaling pathway.

BACKGROUND: Acute kidney injury (AKI) is a serious pathology that causes dysfunction in concentrating urine due to kidney damage, resulting in blood pressure dysregulation and increased levels of toxic metabolites. Dexpanthenol (DEX), a pantothenic acid analog, exhibits anti-inflammatory and anti-apoptotic properties in various tissues. This study investigated the protective effects of DEX against systemic inflammation-induced AKI. METHODS: Thirty-two female rats were randomly assigned to the control, lipopolysaccharide (LPS), LPS+DEX, and DEX groups. LPS (5 mg/kg, single dose on the third day, 6 h before sacrifice) and DEX (500 mg/kg/d for 3 d) were administered intraperitoneally. After sacrifice, blood samples and kidney tissues were collected. Hematoxylin and eosin, caspase-3 (Cas-3), and tumor necrosis factor alpha (TNF-α) staining were performed on the kidney tissues. The total oxidant status (TOS) and total antioxidant status were measured using spectrophotometric methods. Aquaporin-2 (AQP-2), silent information regulator 1 (SIRT1), and interleukin-6 (IL-6) were detected using quantitative reverse transcription-polymerase chain reaction analysis. RESULTS: Histopathological analysis revealed that DEX treatment ameliorated histopathological changes. In the LPS group, an increase in the blood urea nitrogen, creatinine, urea, IL-6, Cas-3, TNF-α, and TOS levels and oxidative stress index was observed compared with the control group, whereas AQP-2 and SIRT1 levels decreased. DEX treatment reversed these effects. CONCLUSIONS: DEX was found to effectively prevent inflammation, oxidative stress, and apoptosis in the kidneys via the SIRT1 signaling pathway. These protective properties suggest DEX's potential as a therapeutic agent for the treatment of kidney pathologies.

Potent carotenoid astaxanthin expands the anti-cancer activity of cisplatin in human prostate cancer cells.

Prostate cancer (PCa) is the second most common type of cancer and the sixth cause of death in men worldwide. Radiotherapy and immunotherapy are commonly used in treating PCa, but understanding the crosstalk mechanisms of carcinogenesis and new therapeutic approaches is essential for supporting poor diagnosis and existing therapies. Astaxanthin (ASX) is a member of the xanthophyll family that is an oxygenated derivative of carotenoids whose synthesis is in plant extracts from lycopene. ASX has protective effects on various diseases, such as Parkinson's disease and cancer by showing potent antioxidant and anti-inflammatory properties. However, there is an ongoing need for a detailed investigation of the molecular mechanism of action to expand its therapeutic use. In the present study, we showed the new regulatory role of ASX in PCa cells by affecting the unfolded protein response (UPR) signaling, autophagic activity, epithelial-mesenchymal transition (EMT) and regulating the expression level of angiogenesis-related protein vascular endothelial growth factor A (VEGF-A), proto-oncogene c-Myc and prostate-specific antigen (PSA). Additionally, we determined that it exhibited synergistic action with cisplatin and significantly enhanced apoptotic cell death in PCa cells. Present findings suggest that ASX may be a potent adjuvant therapeutic option in PCa treatment when used alone or combined with chemotherapeutics. Schematic illustration of the biochemical activity of astaxanthin and its combination with cisplatin.

Circadian Oscillation Pattern of Endoplasmic Reticulum Quality Control (ERQC) Components in Human Embryonic Kidney HEK293 Cells.

The circadian clock regulates the "push-pull" of the molecular signaling mechanisms that arrange the rhythmic organization of the physiology to maintain cellular homeostasis. In mammals, molecular clock genes tightly arrange cellular rhythmicity. It has been shown that this circadian clock optimizes various biological processes, including the cell cycle and autophagy. Hence, we explored the dynamic crosstalks between the circadian rhythm and endoplasmic reticulum (ER)-quality control (ERQC) mechanisms. ER-associated degradation (ERAD) is one of the most important parts of the ERQC system and is an elaborate surveillance system that eliminates misfolded proteins. It regulates the steady-state levels of several physiologically crucial proteins, such as 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) and the metastasis suppressor KAI1/CD82. However, the circadian oscillation of ERQC members and their roles in cellular rhythmicity requires further investigation. In the present study, we provided a thorough investigation of the circadian rhythmicity of the fifteen crucial ERQC members, including gp78, Hrd1, p97/VCP, SVIP, Derlin1, Ufd1, Npl4, EDEM1, OS9, XTP3B, Sel1L, Ufd2, YOD1, VCIP135 and FAM8A1 in HEK293 cells. We found that mRNA and protein accumulation of the ubiquitin conjugation, binding and processing factors, retrotranslocation-dislocation, substrate recognition and targeting components of ERQC exhibit oscillation under the control of the circadian clock. Moreover, we found that Hrd1 and gp78 have a possible regulatory function on Bmal1 turnover. The findings of the current study indicated that the expression level of ERQC components is fine-tuned by the circadian clock and major ERAD E3 ligases, Hrd1 and gp78, may influence the regulation of circadian oscillation by modulation of Bmal1 stability.

Dexamethasone-stimulated glucocorticoid receptor signaling positively regulates the endoplasmic reticulum-associated degradation (ERAD) mechanism in hepatocellular carcinoma cells.

Hepatocellular carcinoma is one of the most common types of primary liver cancer in adults and also it is the third leading cause of cancer-related deaths worldwide. Although there are various treatment options such as surgery, radiation, targeted drug therapy, immunotherapy and chemotherapy, most hepatocellular carcinomas are highly resistant to systemic treatments. Today, the molecular pathogenesis of hepatocellular carcinoma remains largely obscure. Therefore, there is a need for detailed research for the characterization of molecular signaling networks related to the development of hepatocellular carcinoma. Recent studies have attention to the hormonal regulation of hepatocellular carcinoma cells mediated by systemic hormones such as glucocorticoids. However, glucocorticoid-mediated regulation of endoplasmic reticulum-associated degradation (ERAD) and unfolded protein response (UPR), which are known to be important survival mechanisms for cancer cells remains unknown in hepatocellular carcinoma. In the present study, we showed that dexamethasone-induced glucocorticoid receptor signaling mediated advanced regulation of ERAD and UPR signaling in hepatocellular carcinoma cells. Our findings indicated that glucocorticoid signaling positively regulated mRNA and protein levels of ERAD components and also protein kinase RNA-like ER Kinase (PERK) and inositol-requiring enzyme 1⍺ (IRE1⍺) branches of UPR signaling are accompanied by the glucocorticoid signaling. In addition, putative glucocorticoid response elements (GREs) were determined in the promoter regions of ERAD members in in-silico analyses. Additionally, silencing of ERAD components significantly reduced the tumorigenic features of hepatocellular carcinoma cells, including cell proliferation, metastasis, invasion and 3D tumor formation. Collectively, these results reveal a novel pattern of regulation of ERAD components by glucocorticoid-mediated in human hepatocellular carcinoma cells.

Estrogens drive the endoplasmic reticulum-associated degradation and promote proto-oncogene c-Myc expression in prostate cancer cells by androgen receptor/estrogen receptor signaling.

The tumorigenic properties of prostate cancer are regulated by advanced hormonal regulation-mediated complex molecular signals. Therefore, characterizing the regulation of these signal transduction systems is crucial for understanding prostate cancer biology. Recent studies have shown that endoplasmic reticulum (ER)-localized protein quality control mechanisms, including ER-associated degradation (ERAD) and unfolded protein response (UPR) signaling contribute to prostate carcinogenesis and to the development of drug resistance. It has also been determined that these systems are tightly regulated by androgens. However, the role of estrogenic signaling in prostate cancer and its effects on protein quality control mechanisms is not fully understood. Herein, we investigated the regulatory effects of estrogens on ERAD and UPR and their impacts on prostate carcinogenesis. We found that estrogens strongly regulated the ERAD components and IRE1⍺ branch of UPR by Er⍺/ß/AR axis. Besides, estrogenic signaling rigorously regulated the tumorigenicity of prostate cancer cells by promoting c-Myc expression and epithelial-mesenchymal transition (EMT). Moreover, estrogenic signal blockage significantly decreased the tumorigenic features of prostate cancer cells. Additionally, simultaneous inhibition of androgenic/estrogenic signals more efficiently inhibited tumorigenicity of prostate cancer cells, including proliferation, migration, invasion and colonial growth. Furthermore, computational-based molecular docking, molecular dynamics simulations and MMPBSA calculations supported the estrogenic stimulation of AR. Present findings suggested that ERAD components and IRE1⍺ signaling are tightly regulated by estrogen-stimulated AR and Er⍺/ß. Our data suggest that treatment approaches targeting the co-inhibition of androgenic/estrogenic signals may pave the way for new treatment approaches to be developed for prostate cancer. The present model of the impact of estrogens on ERAD and UPR signaling in androgen-sensitive prostate cancer cells.

Progesterone regulates the endoplasmic reticulum-associated degradation and Unfolded Protein Response axis by mimicking the androgenic stimulation in prostate cancer cells.

BACKGROUND: Today, androgen receptor (AR)-mediated signaling mechanisms in prostate cancer are intensively studied. However, the roles of other steroid hormones in prostate cancer and their effects on androgenic signaling still remain a mystery. Recent studies focused on the androgen-mediated regulation of protein quality control mechanisms such as endoplasmic reticulum-associated degradation (ERAD) and unfolded protein response (UPR) in prostate cancer cells. Present study, we investigated the action of progesterone signaling on ERAD and UPR mechanisms and analyzed the crosstalk of progesterone signaling with androgenic signal in prostate cancer cells. METHODS AND RESULTS: The mode of action of progesterone on ERAD, UPR and AR signaling in prostate cancer was investigated by cell culture studies using LNCaP and 22Rv1 cells. To this aim qRT-PCR, western-blotting assay, immunofluorescent microscopy, nuclear fractionation and bioinformatic analysis were used. Our results indicated that progesterone positively regulates mRNA and protein levels of ERAD components in LNCaP cells. Also, it induced the IRE⍺ and PERK branches of UPR signaling. Progesterone receptor antagonist effectively antagonized the progesterone-induced responses. We also had similar results in 22Rv1 cells. Also, we tested the effect of the pharmacologically reducing of IRE⍺ and PERK signaling on progesterone-induced ERAD. Additionally, we determined the presence of putative progesterone response elements (PREs) in the promoter regions of ERAD members by bioinformatic tool. More strikingly, we found progesterone regulates AR signaling by modulating the nuclear transactivation of AR. CONCLUSION: Herein, we defined that progesterone hormone positively regulates ERAD and UPR mechanisms in prostate cancer cells and that progesterone contributes to the molecular biology of prostate cancer by regulating androgenic signaling. Mode of Action of Progesteron on Androgen sensitive prostate cancer cells.

1,25(OH)2 D3 induced vitamin D receptor signaling negatively regulates endoplasmic reticulum-associated degradation (ERAD) and androgen receptor signaling in human prostate cancer cells.

Steroid hormone signaling is critical in the tumor progression and the regulation of physiological mechanisms such as endoplasmic reticulum-associated degradation (ERAD) and unfolded protein response (UPR) in prostate cancer. 1,25(OH)2 D3 is an active metabolite of vitamin D classified as a steroid hormone. It exhibits anti-tumor effects, including angiogenesis and suppression of cell cycle progression. Moreover, progressively reducing expression levels of vitamin D receptor (VDR) are observed in many cancer types, including the prostate. In the present study, we investigated the molecular action of 1,25(OH)2 D3 on ERAD, UPR and androgenic signaling. We found that 1,25(OH)2 D3 negatively regulated the expression level of ERAD components and divergently controlled the inositol-requiring enzyme 1⍺ (IRE1⍺) and protein kinase RNA-like ER kinase (PERK) branches of UPR in LNCaP human prostate cancer cells. Also, similar results were obtained with another human prostate cancer cell line, 22Rv1. More strikingly, we found that androgenic signaling is negatively regulated by VDR signaling. Also, molecular docking supported the inhibitory effect of 1,25(OH)2 D3 on AR signaling. Moreover, we found VDR signaling suppressed tumor progression by decreasing c-Myc expression and reducing the epithelial-mesenchymal transition (EMT). Additionally, 1,25(OH)2 D3 treatment significantly inhibited the 3D-tumor formation of LNCaP cells. Our results suggest that further molecular characterization of the action of VDR signaling in other cancer types such as estrogenic signal in breast cancer will provide important contributions to a better understanding of the roles of steroid hormone receptors in carcinogenesis processes.

Irbesartan decreased mitochondrial stress related apoptosis in cisplatin induced acute kidney injury via regulating BCL-2/BAX signaling.

BACKGROUND: Cisplatin (CPN) is used in the treatment of various cancers. However, the especially nephrotoxic effect is limiting its use. We aimed to evaluate the renoprotective effects of Irbesartan (IBN) on CPN-induced acute kidney injury via mitochondrial stress related apoptosis. METHODS AND RESULTS: 32 rats were divided into 4 groups as control, CPN, CPN + IBN and IBN. Water or IBN 50 mg/kg (orally) was administered for 7 days and a single dose of CPN (5 mg/kg) intraperitoneally was given CPN and CPN + IBN groups on fourth day of experiment. At the end of the experiment, serum BUN and creatinine (Cre) levels, which are the indicators of kidney function are measured. Bcl-2-associated X protein (Bax) and B-cell-lymphoma-2 (Bcl-2) mRNA levels were analyzed by using qRT-PCR from kidneys as a mitochondrial stress indicator. Also, active caspase-3(cas-3) protein and tumor necrosis factor alpha (TNF-α) expressions were examined by immunostaining of the kidney tissues. For evaluation of oxidative stress, malondialdehyde (MDA), total oxidant status (TOS) and total antioxidant status (TAS) levels of renal tissues were measured and oxidative stress index (OSI) were calculated. CPN increased serum BUN and creatinine levels. Also, MDA, TOS and OSI levels were significantly elevated and TAS levels decreased in the CPN group. Moreover, CPN elevated the levels of Bax, active cas-3 protein and TNF-α expressions and suppressed Bcl-2 levels. IBN treatment reversed all these changes. CONCLUSIONS: IBN significantly regressed kidney damage by its anti-inflammatory and antioxidant activity via inhibiting mitochondrial stress. IBN could be used as a renoprotective agent in CPN-induced kidney injury.

A study on Wistar Albino rats: investigating protective role of ramelteon on liver damage caused by methotrexate.

Methotrexate is an important immunosuppressive and antineoplastic drug and is widely used for treatment. However, hepatotoxicity is one of the major adverse effects of methotrexate. In this study, it was aimed to investigate whether ramelteon has a possible protective effect on hepatotoxicity induced by methotrexate. Thirty-two Wistar albino rats were equally divided into four groups: control, methotrexate, methotrexate + ramelteon, and ramelteon. Following a single dose of 20 mg/kg, methotrexate (i.p.), either saline or ramelteon 10 mg/kg (orally) was administered for 7 days. After treatment, animals were sacrificed, and histopathological analyses were evaluated with Hematoxylin-eosin (H-E), immunohistological analyses were evaluated with Interleukin-1 Beta (IL-1ß) and Caspase 3 (CAS-3), biochemical analyzes were evaluated with Total Oxidant Status (TOS), Total antioxidants status (TAS), Oxidative Stress Index (OSI), aspartate aminotransferase (AST), alanine aminotransferase (ALT) activities, at last genetical analyses were evaluated with Sirtuin-1 (SIRT-1) - P53 gene expressions. In the control and ramelteon groups, normal histological structures were observed, while histopathological findings were observed in the methotrexate group. Increasing levels of IL-1ß staining, CAS-3 staining, p53 gene expression, TOS, OSI, AST and ALT were observed in methotrexate group while were observed decreasing levels of TAS and SIRT-1 gene expression (p < 0.05). However, ramelteon reduced the increased findings in methotrexate-induced hepatotoxicity (p < 0.05). The results of the present study showed that ramelteon protects against methotrexate induced hepatotoxicity in rats via SIRT-1 signaling by histological, immunohistological, biochemical and genetical analyses.