The Effects of Remdesivir and Dexamethasone on Renal Sirtuin-1 Expression and Renal Function in Male Rats.

Remdesivir (REM) and dexamethasone (DEX) both have been used to treat coronavirus disease 2019 (COVID-19). The present study aimed to evaluate the effects of REM and DEX on kidney structure and function with particular focus on the probable renal sirtuin-1 (SIRT1) expression alteration in rats. Twenty-four male Wistar rats were divided into four groups, as follows: group A (control) received normal saline (5 mL/kg/day for 10 days); group B (REM) received REM (17 mg/kg/day on the first day, and 8.5 mg/kg/day on the 2nd-10th days); group C (REM + DEX) received both REM (17 mg/kg/day on the first day, and 8.5 mg/kg/day on the 2nd-10th days) and DEX (7 mg/kg/day, for 10 days); group D (DEX) received DEX (7 mg/kg/day for 10 days). Renal SIRT1 expression and kidney structure and function-related factors were evaluated by standard methods. The mean levels of urea in the REM + DEX group (60.83 ± 6.77, mg/dL) were significantly higher than in the control (48.33 ± 3.01, mg/dL; p = 0.002) and DEX (51.22 ± 4.99, mg/dL; p = 0.018) groups. The mean levels of creatinine in the REM (0.48 ± 0.08, mg/dL) and REM + DEX (0.50 ± 0.04, mg/dL) groups were higher than in the control group (48.33 ± 3.0 mg/dL) significantly (p = 0.022 and p = 0.010, respectively). The renal SIRT1 expression was significantly (p = 0.018) lower in the REM + DEX group (0.36 ± 0.35) than in the control group (1.34 ± 0.48). Tubulointerstitial damage (TID) scores in REM + DEX-treated rats (2.60 ± 0.24) were significantly higher than in the control (0.17 ± 0.17, p = 0.001) and DEX (0.50 ± 0.29, p = 0.005) groups. The administration of DEX and REM might lead to kidney injury associated with SIRT1 downregulation.

ctDNA as a novel and promising approach for cancer diagnosis: a focus on hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most prevalent forms of cancer worldwide. Therefore, it is essential to diagnose and treat HCC patients promptly. As a novel discovery, circulating tumor DNA (ctDNA) can be used to analyze the tumor type and the cancer location. Additionally, ctDNA assists the cancer stage determination, which enables medical professionals to provide patients with the most appropriate treatment. This review will discuss the HCC-related mutated genes diagnosed by ctDNA. In addition, we will introduce the different and the most appropriate ctDNA diagnosis approaches based on the facilities.

Enhanced chemotherapeutic efficacy of docetaxel in human lung cancer cell line via GLUT1 inhibitor.

The dose-dependent adverse effects of anticancer agents need new methods with lesser toxicity. The objective of the current research was to evaluate the efficacy of GLUT1 inhibitor, as an inhibitor of glucose consumption in cancer cells, in augmenting the efficiency of docetaxel with respect to cytotoxicity and apoptosis. Cell cytotoxicity was assessed by using methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. Annexin V/PI double staining was employed to evaluate apoptosis percentage. Quantitative real-time polymerase chain reaction (RT-PCR) analysis was accomplished to detect the expression of genes involved in the apoptosis pathway. The IC50 values for docetaxel and BAY-876 were 3.7 ± 0.81 and 34.1 ± 3.4 nM, respectively. The severity of synergistic mutual effects of these agents on each other was calculated by synergy finder application. It showed that the percentage of apoptotic cells following co-administration of docetaxel and BAY-876 increased to 48.1 ± 2.8%. In comparison without GLUT1 co-administration, the combined therapy decreased significantly the transcriptome levels of the Bcl-2 and Ki-67 and a remarkable increase in the level of the Bax as proapoptotic protein(p < 0.05). Co-treatment of BAY-876 and docetaxel depicted a synergistic effect which was calculated using the synergy finder highest single agent (HSA) method (HSA synergy score: 28.055). These findings recommend that the combination of GLUT-1 inhibitor and docetaxel can be considered as a promising therapeutic approach for the treatment of patients with lung cancer.

Zoledronic acid-loaded lipidic nanoparticles enhance apoptosis and attenuate invasiveness by inhibiting epithelial to mesenchymal transition (EMT) in HepG2 cancer cells.

The aim of this study was to evaluate the potential of zoledronic acid (ZOL)-loaded lipidic nanoparticles (ZOL-NLCs) in enhancing the efficiency of paclitaxel (Pac) in the context of cytotoxicity, apoptosis, and invasiveness of HepG2 hepatocellular carcinoma cells. ZOL-NLCs were characterized in terms of zeta potential, particle size, and scanning electron microscope (SEM) as well as cell internalization. To measure the anti-proliferative effects of ZOL-NLCs, annexin-V/PI and MTT assays were employed. Real-time PCR and western blot analysis were performed to identify the molecular mechanisms underlying the apoptosis in response to the studied conditions. Furthermore, the transwell migration assay was applied to clarify the role of applied formulations on the invasiveness of HepG2 cells. Our results demonstrated that the optimized ZOL had an average particle size of 105 ± 6 nm with a nearly narrow size distribution. The IC50 values for ZOL and ZOL-NLCs were 90 ± 3.1 and 54.6 ± 2.4 µM, respectively. The population of apoptotic cells was increased from 17 ± 2% to 27 ± 4% (p < 0.05) in response to treatment with ZOL-NLCs. ZOL-loaded nanoparticles triggered the mRNA expression of Bax as pro-apoptotic marker and E-cadherin as epithelial one along with a decrease in mesenchymal marker, N-cadherin, and Bcl-xl as an anti-apoptotic marker in HepG2 cells. These outcomes were consistent with western blot analysis of protein expressions. Besides, ZOL-incorporated lipidic nanoparticles reduced the migration of HepG2 cells significantly. Our data suggest that the formulation of ZOL into lipidic nanoparticles can be considered a potential therapeutic approach that can enhance the efficacy of Pac chemotherapy.

Sensitization of A-549 lung cancer cells to Cisplatin by Quinacrine-loaded lipidic nanoparticles via suppressing Nrf2 mediated defense mechanism.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is believed to be responsible for the control mechanisms of cellular defense response and master regulator of antioxidant system by adjustment of endogenous antioxidants, phase II detoxifying enzymes and transporters, so inhibition of Nrf2 could be considered molecule target to overcome drug resistance and cancer progression. By harnessing liposome as an advanced nanoparticles transporter, we formulated Quinacrine known as nrf2 inhibitor into nano-carrier, and sensitized A-549 lung tumor cells to Cisplatin. The aim of this work was to prepare liposome nano-carriers to enhance the bioavailability of Quinacrine and to improve passive targeting in A549 cells. Quinacrine formulation into liposome exposed a mean particle size of 80±5 nm in passive targeting and 110±3 after decoration with chitosan oligosaccharides (COS), respectively. The highest amount of cell death (p<0.05) occurred with the co-incubation of the A549 cells with new formulation and Cisplatin. Additionally, Quinacrine-loaded liposomes declined Nrf2 expression more than Quinacrine alone (p<0.05). Correspondingly, the expression of Nrf2 downstream genes, MRP1, Trx, and bcl2 decreased significantly. Taking all the data into consideration, liposomes containing Quinacrine could ameliorate the effectiveness of Cisplatin by raising the permeability of cancer cells to the abovementioned chemical treatment and might be then given as a candidate to boost the therapeutic protocols in cancer patients.

Formulation of Stattic as STAT3 inhibitor in nanostructured lipid carriers (NLCs) enhances efficacy of doxorubicin in melanoma cancer cells.

Nowadays, nanoparticle-based combination therapy has been emerging as huge innovation in cancer treatment. Here, we studied the effect of Stattic (STAT3 inhibitor) loaded in nanostructured lipid carriers (NLCs) on enhancing the efficacy, cytotoxicity, and induction of apoptosis of doxorubicin in B16F10 mouse melanoma cancer cell. The evaluation of Stattic-loaded NLCs has been done in terms of zeta potential, particle size, scanning electron microscope (SEM), and cellular uptake. MTT assay was applied to evaluate the cell proliferation. Apoptotic cell death and identification of early and late apoptosis were assessed by DAPI staining and Annexin V/PI staining, respectively. Real-time RT-PCR was applied to measure the effects of doxorubicin and/or Stattic on key apoptotic genes such as Bad, Survivin, HIF1, and STAT3. The Stattic formulated into NLCs shown mean particle size of 56 ± 7 nm which was confirmed by SEM. The IC50 values for Stattic and doxorubicin were 2.95 ± 0.52 µM and 1.21 ± 0.36 µM, respectively. Stattic-loaded NLCs diminished percent of cell proliferation from 68 ± 6.8 to 54 ± 3.7% (p < 0.05). Combinational treatment of the cells with Stattic-loaded nanoparticles and doxorubicin give rise to a significant increase in the percentage of apoptosis (p < 0.05). The study of gene expression profile has shown a remarkable decrease in anti-apoptotic gene, Survivin, along with smooth decline in HIF1 as angiogenesis intermediator and increase in Bad mRNA levels. Our results recommend that NLCs as novel technology have potent strategy to augment efficacy of current chemotherapeutic agent in melanoma cancer cells.

Identification of Nrf2/STAT3 axis in induction of apoptosis through sub-G 1 cell cycle arrest mechanism in HT-29 colon cancer cells.

We investigated the role of stattic as an adjuvant molecule to increase the cytotoxicity of 5-fluorouracil (5-FU) through specific inhibition of molecular targets, signal transducer and activator of transcription 3 (STAT3) and nuclear factor erythroid 2-related factor 2 (Nrf2) in HT-29 colon cancer cells. Cytotoxicity and apoptotic effects were investigated by methylthiazolyldiphenyl-​tetrazolium bromide assay and flow cytometry analysis, respectively. Real-time polymerase chain reaction was applied to assess the messenger RNA (mRNA) level of STAT3, Nrf2, and apoptotic genes including Bax, Bcl-xl, and Bcl-2. The antitumor effect of 5-FU in combination with stattic induced synergistic effect in HT-29 cells with combination indexes (CIs) 0.49. Flow cytometric results related to apoptotic confirmed that there was up to 40% increase in the population of apoptotic cells in HT-29 colon cancer cells incubated with 5-FU and stattic compared with control groups. Our data from gene expression determined a substantial diminish in the mRNA levels of the Nrf2 and antiapoptotic gene Bcl-2 along with a noticeable increase in the level of the proapoptotic Bax in HT-29 colon cells that underwent cotreatment with 5-FU and stattic (P < 0.05). Moreover, the results exhibited that stattic can be used as adjuvant chemotherapy besides the 5-FU. This therapeutic approach in colon cancer could mediate 5-FU chemoresistance via modulating therapeutic targets (ie, STAT3 and Nrf2 pathways) and decreased 5-FU-related adverse effects.

Adjuvant therapy with stattic enriches the anti-proliferative effect of doxorubicin in human ZR-75-1 breast cancer cells via arresting cell cycle and inducing apoptosis.

Adjuvant therapy with novel and effective component has been presented as a contrivance in breast cancer treatment versus the conventional methods. The current research was done to evaluate the implement of stattic, specific STAT3 inhibitor on the anti-proliferative and apoptotic behavior of doxorubicin on ZR-75-1 breast cancer cells. Cell viability was investigated by MTT assay, the percentage of apoptosis by DAPI staining, and Annexin V. Real Time-PCR was applied to find out the correlation between mechanistic roles of the STAT3 pathway and apoptotic signal in the modulation of Bcl-2 and Bax gene expressions axis. The IC50 values for doxorubicin and stattic were 2.5 ± 0.18 µM and 3.5 ± 0.28 µM, respectively. Combination index (CI) value for ZR-75-1 breast cancer was 0.72, which indicated a strong synergistic effect. Incubation of the cells with a combination of stattic and doxorubicin revealed a significant increase in growth inhibitory effect of doxorubicin with more than 50% decrease in proliferation rate and a two-fold increase in the percentage of apoptotic cells. Assessment of gene expression levels demonstrated a visible decrease in antiapoptotic Bcl-2 and Bcl-xl accompanied by an increase in pro-apoptotic Bax mRNA levels (p < 0.05). Taken together, our results show that combination of a STAT3 inhibitor and doxorubicin can be figured out as a promising approach for dealing of patients with breast cancers.

Fabrication of all-trans-retinoic acid-loaded biocompatible precirol: A strategy for escaping dose-dependent side effects of doxorubicin.

BACKGROUND: Drug delivery-based nanoparticles have been emerged to be an alternative and efficient approach to cancer therapy compared to conventional systems. Here, we investigated the role of all-trans retinoic acid (ATRA) formulated with precirol in increasing doxorubicin (Dox) induced apoptosis and cell cycle arrest in MDA-MB-231 breast cancer cells. METHODS: ATRA-loaded Nano structured lipid carriers (NLCs) were evaluated in terms of particle size, zeta potential, Fourier transforms infrared spectroscopy (FTIR), cell internalization, and scanning electron microscope (SEM). To understand molecular mechanism of apoptosis and cell cycle progression flow cytometric assay, MTT and DAPI staining was applied. Real time (RT)-PCR analysis was employed to investigate the expression of apoptosis related genes, including Survivin, Bcl-2 and Bax. RESULTS: The optimized ATRA formulation exhibited average particle size of 95±5nm with nearly narrow size distribution. The IC50 values for ATRA and doxorubicin were 48±0.4µM and 0.81±0.02µM, respectively. ATRA-loaded NLCs decreased percentage of cell proliferation from 51±7.2% to 36±4.1% (p <0.05). Co-treatment of the MDA-MB-231 cells with ATRA formulation and doxorubicin caused two-fold increase in the percentage of apoptosis (p<0.05). The results from gene expression exhibited a significant decrease in survivin along with increase at Bax mRNA levels accompanied by a slight increase in Bax/Bcl-2 ratio. CONCLUSION: Our results propose that ATRA encapsulated in precirol as a biocompatible compound augments the efficacy of Dox in cancer therapy.

Vitamin D-Loaded Nanostructured Lipid Carrier (NLC): A New Strategy for Enhancing Efficacy of Doxorubicin in Breast Cancer Treatment.

The dangerous dose-dependent side effects of anticancer agents triggered the finding of new approaches for elevated chemotherapy efficacy. This study investigated the potential application of nanostructured lipid careers (NLCs) in increasing vitamin D3 (VitD) effectiveness in breast cancer cell (MCF-7) in concurrent administration with doxorubicin (Dox). VitD-loaded NLCs were characterized by particle size, zeta potential, Fourier transform infrared spectroscopy, and scanning electron microscope. Cytotoxicity and molecular effects of formulation were evaluated by MTT, DAPI staining, flow cytometry, and real-time quantitative PCR assays. The formulation revealed mean particle size of 87±5 nm with a polydispersity index of 0.24 confirmed by SEM images. The IC50 values for VitD and Dox were 1.3 ± 0.04 and 0.65 ± 0.05 µM, respectively. VitD-loaded NLCs decreased the percentage of cell proliferation from 49 ± 7.2% to 37 ± 5.1% (P < 0.05). Cotreatment of the cells with VitD-loaded NLCs and Dox caused over a twofold increase in the percentage of apoptosis (P < 0.05). Gene expression profile demonstrated a significant decrease in antiapoptotic factor survivin along with increase in proapoptotic factor Bax mRNA levels. Overall, our results introduced the NLC technology as a novel strategy to elevate the efficacy of chemotherapeutics in breast cancer.

Combined Treatment with Stattic and Docetaxel Alters the Bax/Bcl-2 Gene Expression Ratio in Human Prostate Cancer Cells

Docetaxel, recognized as a stabilizing microtubule agent, is frequently administrated as a first line treatment for prostate cancers. Due to high side effects of monotherapy, however, combinations with novel adjuvants have emerged as an alternative strategy in cancer therapy protocols. Here, we investigated the combined effects of stattic and docetaxel on the DU145 prostate cancer cell line. Cytotoxicity was evaluated by MTT assay. To understand molecular mechanisms of stattic action, apoptotic related genes including Bcl-2, Mcl-1, Survivin and Bax were evaluated by real-time RT-PCR. Alteration in the expression of pro-apoptotic Bax and anti-apoptotic Bcl-2 genes and Bax/Bcl-2 ratio were investigated via the 2ΔΔCT method. The IC50 values for docetaxel and stattic were 3.7 ± 0.9 nM and 4.6±0.8 µM, respectively. Evaluation of key gene expression levels revealed a noticeable decrease in antiapoptotic Bcl-2 and Mcl-1 along with an increase in pro-apoptotic Bax mRNA levels (p<0.05). Our results suggest that combination of a STAT3 inhibitor with doctaxel can be considered as a potent strategy for induction of apoptosis via increasing Bax mRNA expression.

Sustained release of melatonin: A novel approach in elevating efficacy of tamoxifen in breast cancer treatment.

BACKGROUND: Finding advanced anti-cancer agents with selective toxicity in tumor tissues is the goal of anticancer delivery systems. This study investigated potential application of nanostructured lipid carriers (NLCs) in increasing melatonin induced cytotoxicity and apoptosis in MCF-7 breast cancer cells. METHODS: Melatonin-loaded NLCs were characterized for particle size, zeta potential, Fourier transforms infrared spectroscopy, differential scanning calorimetry, cellular uptake, and scanning electron microscope (SEM). Anti-proliferative and apoptotic effects of new formulation were evaluated by MTT and flow cytometric assays, respectively. Gene expression of apoptotic markers including survivin, Bcl-2 and Bid were examined by Real time quantitative PCR. RESULTS: The optimized formulation of NLCs revealed mean particle size of 71±5nm with nearly narrow size distribution. The formulation exhibited an acceptable stability during four months in terms of size and lack of drug release. The IC50 values for melatonin and tamoxifen were 1.3±0.4mM and 30.7±5.2µM, respectively. Melatonin loaded NLCs decreased percentage of cell proliferation from 55±7.2% to 40±4.1% (p<0.05). Co-treatment of the cells with melatonin loaded nanoparticles and tamoxifen caused two fold increase in the percentage of apoptosis (p<0.05). Evaluation of gene expression profile demonstrated a marked decrease in anti-apoptotic survivin with increase in pro-apoptotic Bid mRNA levels. CONCLUSION: Taken together, our results suggest NLC technology as a promising delivery system, which elevates the efficacy of chemotherapeutics in breast cancer cells.

The role of Six1 signaling in paclitaxel-dependent apoptosis in MCF-7 cell line.

The resistance of cancer cells to chemotherapeutic agents represents the main problem in cancer treatment. Despite intensive research, mechanisms of resistance have not yet been fully elucidated. Six1 signaling has an important role in the expansion of progenitor cell populations during early embryogenesis. Six1 gene overexpression has been strongly associated with aggressiveness, invasiveness, and poor prognosis of different cancers. In this study, we investigated the role of Six1 signaling in resistance of MCF-7 breast cancer cells to taxanes. We first established in vitro paclitaxel-resistant MCF-7 breast cancer cells. Morphological modifications in paclitaxel-resistant cells were examined via light microscopic images and fluorescence-activated cell sorting analysis. Applying quantitative real-time polymerase chain reaction, we measured Six1, B-cell lymphoma/leukemia(BCL-2), BAX, and P53 mRNA expression levels in both non-resistant and resistant cells. Resistant cells were developed from the parent MCF-7 cells by applying increasing concentrations of paclitaxel up to 64 nM. The inhibitory concentration 50% value in resistant cells increased from 3.5 ± 0.03 to 511 ± 10.22 nM (p = 0.015). In paclitaxel-resistant cells, there was a significant increase in Six1 and BCL-2 mRNA levels (p = 0.0007) with a marked decrease in pro-apoptotic Bax mRNA expression level (p = 0.03); however, there was no significant change in P53 expression (p = 0.025). Our results suggest that identifying cancer patients with high Six1 expression and then inhibition of Six1 signaling can improve the efficiency of chemotherapeutic agents in the induction of apoptosis.

Folate-Targeted Nanostructured Lipid Carriers (NLCs) Enhance (Letrozol) Efficacy in MCF-7 Breast Cancer Cells

Objective: Targeted-drug-delivery based lipid nanoparticles has emerged as a new and effective approach in cancer chemotherapy. Here, we investigated the ability of folate-modified nanostructured lipid carriers (NLCs) to enhance letrozol (LTZ) efficacy in MCF-7 breast cancer cells. Methods: New formulations were evaluated regarding to particle size and scanning electron microscope (SEM) features. Anti-proliferative effects of LTZ loaded nanoparticles were examined by MTT assay. To understand molecular mechanisms of apoptosis and cell cycle progression, flow cytometric assays were applied. Results: Optimum size of nanoparticles was obtained in mean average of 98 ± 7 nm with a poly dispersity index (PDI) of 0.165. The IC50 value was achieved for LTZ was 2.2 ± 0.2 µM. Folate-NLC-LTZ increased the percentage of apoptotic cells from 24.6% to 42.2% compared LTZ alone (p<0.05). Furthermore, LTZ loaded folate targeted NLCs caused marked accumulation of cells in the subG1 phase. Conclusion: Taken together, our results concluded that folate targeted LTZ can be considered as potential delivery system which may overcome limitations of clinical application of LTZ and improve drug efficacy in tumor tissue.

Construction of Yeast Recombinant Expression Vector Containing Human Epidermal Growth Factor (hEGF).

PURPOSE: The objective of this study was construction of recombinant hEGF-pPIC9 which may be used for expression of recombinant hEGF in following studies. METHODS: EGF cDNA was purchased from Genecopoeia Company and used for PCR amplification. Prior to ligation, the PCR product and pPIC9 vector was digested with EcoRI and XhoI and ligated in pPIC9 vector and subjected to colony PCR screening and sequencing analysis. RESULTS: PCR amplification of EGF cDNA using recombinant hEGF-pPIC9 vector as template was concluded in amplification of 197bp fragment. Construction of recombinant hEGF-pPIC9 of EGf gene was verified by PCR and sequencing. CONCLUSION: Construction of Recombinant hEGF-pPIC9 was the primary stage for production and expression of EFG in the future study.