Investigation the apoptotic effect of silver nanoparticles (Ag-NPs) on MDA-MB 231 breast cancer epithelial cells via signaling pathways.

Background: The discovery of novel cancer therapeutic strategies leads to the development of nanotechnology-based methods for cancer treatment. Silver nanoparticles (Ag-NPs) have garnered considerable interest owing to their size, shape, and capacity to modify chemical, optical, and photonic properties. This study aimed to investigate the impact of Ag-NPs on inducing of apoptosis in MDA-MB 231 cells by examining specific signaling pathways. Materials and methods: The cytotoxicity of Ag-NPs was determined using an MTT assay in MDA-MB 231 cells. The apoptotic effects were assessed using the Annexin-V/PI assay. Real-time PCR and western blotting were conducted to analyze the expression of apoptosis-related genes and proteins, respectively. Levels of ERK1/2 and cyclin D1 were measured using ELISA. Cell cycle assay was determined by flow cytometry. Cell migration was evaluated by scratch assay. Results: The results revealed that Ag-NPs triggered apoptosis and cell cycle arrest in MDA-MB 231 cells. The expression level of Bax (pro-apoptotic gene) was increased, while Bcl-2 (anti-apoptotic gene) expression was decreased. Increased apoptosis was correlated with increased levels of p53 and PTEN. Additionally, notable alterations were observed in protein expression related to the Janus kinase/Signal transducers (JAK/STAT) pathway, including p-AKT. Additionally, reduced expression of h-TERT was observed following exposure to Ag-NPs. ELISA results demonstrated a significant reduction in p-ERK/Total ERK and cyclin D1 levels in Ag-NPs-exposed MDA-MB 231 cells. Western blotting analysis also confirmed the reduction of p-ERK/Total ERK and cyclin D1. Decreased level of cyclin D is associated with suppression of cell cycle progression. The migratory ability of MDA-MB-231 cells was reduced upon treatment with Ag-NPs. Conclusions: Our findings revealed that Ag-NPs influenced the proliferation, apoptosis, cell cycle, and migration in MDA-MB 231 cells, possibly by modulating protein expression of the AKT/ERK/Cyclin D1 axis.

Nano-delivery of Silibinin Potentiate the Induction of Immunogenic Cell Death (ICD) in Melanoma Cells.

BACKGROUND: Induction of immunogenic cell death (ICD) in tumors can enhance antitumor immunity and modulate immunosuppression in the tumor microenvironment (TME). OBJECTIVE: In the current study, we investigated the effect of silibinin, a natural compound with anticancer activity, and its polymer-based nanoformulations on the induction of apoptosis and ICD in cancer cells. METHODS: Free and nanoparticulate silibinin were evaluated for their growth-inhibitory effects using an MTT assay. Annexin V/PI staining was used to analyze apoptosis. Calreticulin (CRT) expression was measured by flow cytometry. Western blotting was conducted to examine the levels of elf2α, which plays a role in the ICD pathway. The HSP90 and ATP levels were determined using specific detection kits. RESULTS: Compared to the free drug, silibinin-loaded nanocarriers significantly increased the induction of apoptosis and ICD in B16F10 cells. ICD induction was characterized by significantly increased levels of ICD biomarkers, including CRT, HSP90, and ATP. We also observed an increased expression of p-elf-2α/ elf-2α in B16F10 cells treated with silibinin-loaded micelles compared to cells that received free silibinin. CONCLUSION: Our findings showed that the encapsulation of silibinin in polymeric nanocarriers can potentiate the effects of this drug on the induction of apoptosis and ICD in B16F10 melanoma cells.

The synergistic effects of betanin and radiotherapy in a prostate cancer cell line: an in vitro study.

BACKGROUND: Prostate cancer is among the most common cancers in men with an increasing incidence rate. Radiation therapy (RT) is a therapeutic strategy for the management of prostate cancer after surgery; nonetheless, it has different side effects on neighboring healthy cells/tissues. Moreover, radioresistance has been an increasing phenomenon in the recent years. Therefore, there is an urgent need for the introduction of a safe and effective radiosensitizing agent. Accordingly, the recent trend in the development of novel drugs is accompanied by a push toward natural compounds. Our study evaluated the effects of betanin combined with RT as a potential radiosensitizing agent in the PC-3 cell line. METHODS AND RESULTS: MTT assay was utilized to determine the growth inhibitory impact of betanin. The possible synergistic effect was evaluated with CompuSyn software upon Trypan blue exclusion test. Apoptosis-related gene expression was evaluated via Real-time PCR and the protein expression of P21 was determined using western blotting. A synergistic anticancer effect with an optimal combination index of 0.61 was achieved by treating PC-3 cells with betanin and RT. The results pointed out that betanin synergistically triggered RT-mediated apoptosis and cell cycle arrest through modulating gene and protein expression in comparison with each of the monotherapies. CONCLUSION: These findings shed light on the synergistic antitumor effect of betanin and RT in prostate cancer, indicating the potential use of betanin as a radiosensitizer agent.

Stimulators of immunogenic cell death for cancer therapy: focusing on natural compounds.

A growing body of evidence indicates that the anticancer effect of the immune system can be activated by the immunogenic modulation of dying cancer cells. Cancer cell death, as a result of the activation of an immunomodulatory response, is called immunogenic cell death (ICD). This regulated cell death occurs because of increased immunogenicity of cancer cells undergoing ICD. ICD plays a crucial role in stimulating immune system activity in cancer therapy. ICD can therefore be an innovative route to improve anticancer immune responses associated with releasing damage-associated molecular patterns (DAMPs). Several conventional and chemotherapeutics, as well as preclinically investigated compounds from natural sources, possess immunostimulatory properties by ICD induction. Natural compounds have gained much interest in cancer therapy owing to their low toxicity, low cost, and inhibiting cancer cells by interfering with different mechanisms, which are critical in cancer progression. Therefore, identifying natural compounds with ICD-inducing potency presents agents with promising potential in cancer immunotherapy. Naturally derived compounds are believed to act as immunoadjuvants because they elicit cancer stress responses and DAMPs. Acute exposure to DAMP molecules can activate antigen-presenting cells (APCs), such as dendritic cells (DCs), which leads to downstream events by cytotoxic T lymphocytes (CTLs) and natural killer cells (NKs). Natural compounds as inducers of ICD may be an interesting approach to ICD induction; however, parameters that determine whether a compound can be used as an ICD inducer should be elucidated. Here, we aimed to discuss the impact of multiple ICD inducers, mainly focusing on natural agents, including plant-derived, marine molecules, and bacterial-based compounds, on the release of DAMP molecules and the activation of the corresponding signaling cascades triggering immune responses. In addition, the potential of synthetic agents for triggering ICD is also discussed.

Clinical Significance of Carnitine in the Treatment of Cancer: From Traffic to the Regulation.

Metabolic reprogramming is a common hallmark of cancer cells. Cancer cells exhibit metabolic flexibility to maintain high proliferation and survival rates. In other words, adaptation of cellular demand is essential for tumorigenesis, since a diverse supply of nutrients is required to accommodate tumor growth and progression. Diversity of carbon substrates fueling cancer cells indicate metabolic heterogeneity, even in tumors sharing the same clinical diagnosis. In addition to the alteration of glucose and amino acid metabolism in cancer cells, there is evidence that cancer cells can alter lipid metabolism. Some tumors rely on fatty acid oxidation (FAO) as the primary energy source; hence, cancer cells overexpress the enzymes involved in FAO. Carnitine is an essential cofactor in the lipid metabolic pathways. It is crucial in facilitating the transport of long-chain fatty acids into the mitochondria for ß-oxidation. This role and others played by carnitine, especially its antioxidant function in cellular processes, emphasize the fine regulation of carnitine traffic within tissues and subcellular compartments. The biological activity of carnitine is orchestrated by specific membrane transporters that mediate the transfer of carnitine and its derivatives across the cell membrane. The concerted function of carnitine transporters creates a collaborative network that is relevant to metabolic reprogramming in cancer cells. Here, the molecular mechanisms relevant to the role and expression of carnitine transporters are discussed, providing insights into cancer treatment.

Chrysin and chrysin-loaded nanocarriers induced immunogenic cell death on B16 melanoma cells.

Induction of immunogenic cell death (ICD) is a promising strategy for cancer immunotherapy. Chrysin, which has potential anticancer effects, faces limitations in clinical applications due to its poor water solubility. This study aimed to formulate chrysin with PEG-poly(α-benzylcarboxylate-ε-caprolactone) (PBCL) nanoparticles (NPs) and assess their anticancer and ICD-inducing potency in melanoma cells, comparing with free chrysin. The co-solvent evaporation method was employed to develop chrysin-loaded NPs. UV spectroscopy, dynamic light scattering, and the dialysis bag method were used to evaluate the encapsulation efficiency (EE), particle size, polydispersity index (PDI), and drug release profile, respectively. The anticancer effects of the drugs were assessed using the MTT and trypan blue exclusion assays. Flow cytometry was employed to evaluate apoptosis and calreticulin (CRT) expression. ELISA and western blotting were used to detect heat shock protein 90 (HSP90), Annexin A1, GRP78 (Glucose-related protein78), and activated protein kinase R-like endoplasmic reticulum kinase (p-PERK). Chrysin-loaded PEG-PBCL NPs (chrysin-PEG-PBCL) showed an EE of 97 ± 1%. Chrysin-PEG-PBCL was 38.18 ± 3.96 nm in size, with a PDI being 0.62 ± 0.23. Chrysin-PEG-PBCL showed an initial burst release, followed by sustained release over 24 h. Chrysin-PEG-PBCL exhibited a significantly stronger anticancer effect in B16 cells. Chrysin-PEG-PBCL was found to be more potent in inducing apoptosis. Both free chrysin and chrysin NPs induced ICD as indicated by an increase in the levels of ICD biomarkers. Interestingly, chrysin NPs were found to be more potent inducers of ICD than the free drug. These findings demonstrate that chrysin and chrysin-PEG-PBCL NPs can induce ICD in B16 cells. PEG-PBCL NPs significantly enhanced the potency of chrysin in inducing ICD compared to its free form.

Synergistic effect of chrysin and radiotherapy against triple-negative breast cancer (TNBC) cell lines

Purpose Triple-negative breast cancer (TNBC) is the most aggressive form of breast cancer, accounting for 20% of cases. Due to the lack of a molecular target, limited options are available for TNBC treatment. Radiation therapy (RT) is a treatment modality for the management of TNBC following surgery; however, it has a detrimental effect on surrounding healthy tissues/cells at a higher rate. Methods We examined the effect of RT in combination with chrysin as a possible radiosensitizing agent in an MDA-MB-231 cell line as a model of a TNBC. The growth inhibitory effects of chrysin were examined using an MTT assay. Flow cytometry was performed to evaluate apoptosis and expression of hypoxia-induced factor-1α (HIF-1α). The protein expression of p-STAT3/STAT3 and Cyclin D1 was examined using western blotting. Real-time PCR determined apoptotic-related genes (Bax, BCL2, p53). Results Treatment of MDA-MB-231 cells with chrysin in combination with RT caused synergistic antitumor effects, with an optimum combination index (CI) of 0.495. Our results indicated that chrysin synergistically potentiated RT-induced apoptosis in MDA-MB-231 compared with monotherapies (chrysin and/or RT alone). Expression of HIF-1α was decreased in the cells exposed to combinational therapy. The apoptotic effect of combinational therapy was correlated with increased Bax (pro-apoptotic gene) and p53 levels along with reduced expression of Bcl-2 (anti-apoptotic gene). Increased apoptosis was associated with reduced expression of Cyclin D1, p-STAT3. Conclusion These findings highlight the potential effect of chrysin as a radiosensitizer, indicating the synergistic anti-cancer effect of chrysin and RT in TNBC. Further investigation is warranted in this regard (AU)

Synergistic effect of chrysin and radiotherapy against triple-negative breast cancer (TNBC) cell lines.

PURPOSE: Triple-negative breast cancer (TNBC) is the most aggressive form of breast cancer, accounting for 20% of cases. Due to the lack of a molecular target, limited options are available for TNBC treatment. Radiation therapy (RT) is a treatment modality for the management of TNBC following surgery; however, it has a detrimental effect on surrounding healthy tissues/cells at a higher rate. METHODS: We examined the effect of RT in combination with chrysin as a possible radiosensitizing agent in an MDA-MB-231 cell line as a model of a TNBC. The growth inhibitory effects of chrysin were examined using an MTT assay. Flow cytometry was performed to evaluate apoptosis and expression of hypoxia-induced factor-1α (HIF-1α). The protein expression of p-STAT3/STAT3 and Cyclin D1 was examined using western blotting. Real-time PCR determined apoptotic-related genes (Bax, BCL2, p53). RESULTS: Treatment of MDA-MB-231 cells with chrysin in combination with RT caused synergistic antitumor effects, with an optimum combination index (CI) of 0.495. Our results indicated that chrysin synergistically potentiated RT-induced apoptosis in MDA-MB-231 compared with monotherapies (chrysin and/or RT alone). Expression of HIF-1α was decreased in the cells exposed to combinational therapy. The apoptotic effect of combinational therapy was correlated with increased Bax (pro-apoptotic gene) and p53 levels along with reduced expression of Bcl-2 (anti-apoptotic gene). Increased apoptosis was associated with reduced expression of Cyclin D1, p-STAT3. CONCLUSION: These findings highlight the potential effect of chrysin as a radiosensitizer, indicating the synergistic anti-cancer effect of chrysin and RT in TNBC. Further investigation is warranted in this regard.

Silibinin induces immunogenic cell death in cancer cells and enhances the induced immunogenicity by chemotherapy.

Introduction: Silibinin is a natural flavonoid compound known to induce apoptosis in cancer cells. Despite silibinin's safety and efficacy as an anticancer drug, its effects on inducing immunogenic cell death (ICD) are largely unknown. Herein, we have evaluated the stimulating effects of silibinin on ICD in cancer cells treated with silibinin alone or in combination with chemotherapy. Methods: The anticancer effect of silibinin, alone or in combination with doxorubicin or oxaliplatin (OXP), was assessed using the MTT assay. Compusyn software was used to analyze the combination therapy data. Western blotting was conducted to examine the level of STAT3 activity. Flow cytometry was used to analyze calreticulin (CRT) and apoptosis. The heat shock protein (HSP70), high mobility group box protein1 (HMGB1), and IL-12 levels were assessed by ELISA. Results: Compared to the negative control groups, silibinin induced ICD in CT26 and B16F10 cells and significantly enhanced the induction of this type of cell death by doxorubicin, and these changes were allied with substantial increases in the level of damage-associated molecular patterns (DAMPs) including CRT, HSP70, and HMGB1. Furthermore, conditioned media from cancer cells exposed to silibinin and doxorubicin was found to stimulate IL-12 secretion in dendritic cells (DCs), suggesting the link of this treatment with the induction of Th1 response. Silibinin did not augment the ICD response induced by OXP. Conclusion: Our findings showed that silibinin can induce ICD and it potentiates the induction of this type of cell death induced by chemotherapy in cancer cells.

Silibinin-Loaded Nanostructured Lipid Carriers for Growth Inhibition of Cisplatin-Resistant Ovarian Cancer Cells.

Cisplatin is the most often used chemotherapy in the treatment of ovarian cancer (OC), however long-term usage leads to drug resistance and treatment failure. Silibinin is a sparingly water-soluble natural compound with well-known anticancer effects. The use of lipid-based delivery systems is a potential approach for enhancing silibinin's water solubility. In this study, nanostructured lipid carriers (NLCs) containing silibinin were prepared and their inhibitory effects were tested in combination with cisplatin against sensitive/resistant A2780 OC cells. Silibinin-loaded NLCs (silibinin-NLCs) were prepared by the hot homogenization method, and their size, shape, zeta potential (ZP), and encapsulation efficiency (EE), as well as their inhibitory effects, were examined in combination with cisplatin against sensitive/resistant A2780 OC cells. Formulation of silibinin-NLCs using cocoa butter led to spherical-shaped NLCs with a size of 95 nm and EE of 98%. The ZP and the dispersion index of the silibinin-NLCs were -27.12 ± 0.13 mv and 0.12 ± 0.04, respectively. The release kinetics of silibinin-NLCs was best fitted with the zero-order model. The combination of cisplatin and silibinin-NLCs sensitized the cisplatin-resistant A2780 OC cells and exhibited a more synergistic inhibitory effect on A2780 cells as compared with the combination of cisplatin and plain silibinin. The optimized silibinin-NLCs can be considered a suitable drug delivery system for the inhibition of cisplatin-resistant OC cells.

Recent clinical findings on the role of kinase inhibitors in COVID-19 management.

The highly pathogenic, novel coronavirus disease (COVID-19) outbreak has emerged as a once-in-a-century pandemic with poor consequences, urgently calling for new therapeutics, cures, and supportive interventions. It has already affected over 250 million people worldwide; thereby, there is a need for novel therapies to alleviate the related complications. There is a paradigm shift in developing drugs and clinical practices to combat COVID-19. Several clinical trials have been performed or are testing diverse pharmacological interventions to alleviate viral load and complications such as cytokine release storm (CRS). Kinase-inhibitors have appeared as potential antiviral agents for COVID-19 patients due to their efficacy against CRS. Combination of kinase inhibitors with other therapies can achieve more efficacy against COVID-19. Based on the pre-clinical trials, kinase inhibitors such as Janus kinase-signal transducer and activator of transcription (JAK/STAT) inhibitors, Brutton's tyrosin kinase (BTK) inhibitors, p38 mitogen-activated protein kinases (p38 MAPK) inhibitors, Glycogen synthase kinase 3 (GSK-3) inhibitors can be a promising strategy against COVID-19. Kinase inhibitors possess crucial pharmacological properties for a successful re-purposing in terms of dual anti-inflammatory and anti-viral effects. This review will address the current clinical evidence and the newest discovery regarding the application of kinase inhibitors in COVID-19. An outlook on ongoing clinical trials (clinicaltrials.gov) and unpublished data is also presented here. Besides, Kinase inhibitors' function on COVID-19-mediated CRS is discussed.

Efficient process development for high-level production, purification, formulation, and characterization of recombinant mecasermin in Escherichia coli.

Overproduction of recombinant mecasermin was achieved by investigation of effect of three factors, temperature, inducer amount, and culture media, at three levels according to the Taguchi statistical design in Escherichia coli in a bench-scale bioreactor. In optimal conditions (induction temperature 28 °C, terrific broth with glucose (TB+G) medium, with 0.1 mM IPTG as inducer) 0.84 g/L mecasermin with expression levels of 38% of total protein and 4.13 g/L final dry cell biomass was produced, that is one of the highest values of recombinant protein has been reported in the batch system. The cell disruption was done by lysozyme pretreatment with sonication to the efficient purification of mecasermin. The isolated and washed inclusion bodies were solubilized in Gdn-HCl at pH 5.4 and folded with glutathione and purified with gel filtration. The purified rhIGF-1 (mecasermin) was formulated with arginine. Mecasermin protein remained t stable at 4 °C for up to 2 years. The quantitative and qualitative control indicated that mecasermin is expressed correctly (without the initial methionine by mass spectrometry), pure (without endotoxin and other protein impurities), correct folding (FTIR, RF-HPLC), monomer form (SEC-HPLC), and active (bioactivity test). Also, the purification results revealed that expression at low temperature results in the efficient purification of the overproduced mecasermin with high quantity and quality.

Biomedical Applications of TiO2 Nanostructures: Recent Advances.

Titanium dioxide (TiO2) nanostructures are one of the most plentiful compounds that have emerged in various fields of technology such as medicine, energy and biosensing. Various TiO2 nanostructures (nanotubes [NTs] and nanowires) have been employed in photoelectrochemical (PEC) biosensing applications, greatly enhancing the detection of targets. TiO2 nanostructures, used as reinforced material or coatings for the bare surface of titanium implants, are excellent additive materials to compensate titanium implants deficiencies-like poor surface interaction with surrounding tissues-by providing nanoporous surfaces and hierarchical structures. These nanostructures can also be loaded by diversified drugs-like osteoporosis drugs, anticancer and antibiotics-and used as local drug delivery systems. Furthermore, TiO2 nanostructures and their derivatives are new emerging antimicrobial agents to overcome human pathogenic microorganisms. However, like all other nanomaterials, toxicity and biocompatibility of TiO2 nanostructures must be considered. This review highlights recent advances, along with the properties and numerous applications of TiO2-based nanostructure compounds in nano biosensing, medical implants, drug delivery and antibacterial fields. Moreover, in the present study, some recent advances accomplished on the pharmaceutical applications of TiO2 nanostructures, as well as its toxicity and biocompatibility, are presented.

Poly(ethylene glycol)-poly(ε-caprolactone)-based micelles for solubilization and tumor-targeted delivery of silibinin.

Introduction: Silibinin is a naturally occurring compound with known positive impacts on prevention and treatment of many types of human illnesses in general and cancer in particular. Silibinin is poorly water soluble which results in its insufficient bioavailability and lack of therapeutic efficacy in cancer. Here, we proposed to examine the potential of micelles composed of poly(ethylene glycol) (PEG) as the hydrophilic block and poly(ε-caprolactone) (PCL), poly(α-benzylcarboxylate-ε-caprolactone) (PBCL), or poly(lactide)-(PBCL) (PLA-PBCL) as hydrophobic blocks for enhancing the water solubility of silibinin and its targeted delivery to tumor. Methods: Co-solvent evaporation method was used to incorporate silibinin into PEG-PCL based micelles. Drug release profiles were assessed using dialysis bag method. MTT assay also was used to analyze functional activity of drug delivery in B16 melanoma cells. Results: Silibinin encapsulated micelles were shown to be less than 60 nm in size. Among different structures under study, the one with PEG-PBCL could incorporate silibinin with the highest encapsulation efficiency being 95.5%, on average. PEG-PBCL micelles could solubilize 1 mg silibinin in 1 mL water while the soluble amount of silibinin was found to be 0.092 mg/mL in the absence of polymeric micelles. PEG-PBCL micelles provided the sustained release of silibinin indicated with less than 30% release of silibinin within 24 hours. Silibinin encapsulated in PEG-PBCL micelles resulted in growth inhibitory effect in B16 cancer cells which was significantly higher than what observed with free drug. Conclusion: Our findings showed that PEG-PBCL micellar nanocarriers can be a useful vehicle for solubilization and targeted delivery of silibinin.

Preparation and characterization of PLGA-PEG-PLGA polymeric nanoparticles for co-delivery of 5-Fluorouracil and Chrysin.

Despite significant advances in cancer therapy, chemotherapeutic agents are still the main types of drugs used to treat cancer patients. 5-Fluorouracil (5-FU) is the first-line treatment in several types of human cancers, however, nonspecific function, low plasma half-life, and high doses toxicity are the important barrier to achieve efficient response in cancer patients. The use of polymeric nanoparticles (NPs) for tumor targeted delivery of 5-FU in combination with other potent anticancer agent is considered an important strategy to enhance the therapeutic efficacy of 5-FU. In this study, we proposed to use PLGA-PEG-PLGA NPs to co-encapsulate 5-FU and Chrysin, a natural compound known to enhance the therapeutic efficacy of chemotherapy. NPs were prepared by double emulsion method and characterized for size and drug encapsulation efficacy. The cell growth inhibitory effect of prepared NPs was assessed by MTT assay in HT29 human colon cancer cell line. The analysis of NPs by dynamic light scattering showed that the developed NPs have average size of 40 nm. The encapsulation efficiency of NPs was 81.3% and 97.5% for 5-FU and Chrysin, respectively. Furthermore, the NPs showed a remarkable uptake in HT29 cells. NPs loaded with both 5-FU and Chrysin (5-FU@Chrysin loaded NPs) were found to have significantly higher growth inhibitory effects compared with NPs loaded with each drug alone in HT29 cell line. The synergistic anticancer effects of 5-FU and Chrysin loaded in NPs were confirmed with the combination index (CI) being 0.35. CI for combination therapy with free 5-FU and Chrysin was found to be 0.73, indicating weaker synergistic anticancer effects of these two drugs in free forms as compared with 5-FU@Chrysin loaded NPs. These finding indicates that co-delivery of 5-FU and Chrysin with PLGA-PEG-PLGA copolymer can be used to improve the therapeutic and functional delivery efficacy of 5-FU and Chrysin in cancer.

Clinical application of immune checkpoints in targeted immunotherapy of prostate cancer.

Immunotherapy is considered as an effective method for cancer treatment owing to the induction of specific and long-lasting anti-cancer effects. Immunotherapeutic strategies have shown significant success in human malignancies, particularly in prostate cancer (PCa), a major global health issue regarding its high metastatic rates. In fact, the first cancer vaccine approved by FDA was Provenge, which has been successfully used for treatment of PCa. Despite the remarkable success of cancer immunotherapy in PCa, many of the developed immunotherapy methods show poor therapeutic outcomes. Immunosuppression in tumor microenvironment (TME) induced by non-functional T cells (CD4+ and CD8+), tolerogenic dendritic cells (DCs), and regulatory T cells, has been reported to be the main obstacle to the effectiveness of anti-tumor immune responses induced by an immunotherapy method. The present review particularly focuses on the latest findings of the immune checkpoints (ICPs), including CTLA-4, PD-1, PD-L1, LAG-3, OX40, B7-H3, 4-1BB, VISTA, TIM-3, and ICOS; these checkpoints are able to have immune modulatory effects on the TME of PCa. This paper further discusses different approaches in ICPs targeting therapy and summarizes the latest advances in the clinical application of ICP-targeted therapy as monotherapy or in combination with other cancer therapy modalities in PCa.

STAT3 inhibitory stattic enhances immunogenic cell death induced by chemotherapy in cancer cells.

BACKGROUND: Induction of immunogenic cell death (ICD) is considered a promising strategy for cancer immunotherapy. Stattic is an inhibitor of STAT3, which is found constitutively active in many cancers and plays a major role in cancer progression. OBJECTIVES: In the present study, we proposed to evaluate whether stattic can enhance the effects of chemotherapy in the induction of ICD in cancer cells harboring hyperactive STAT3. METHODS: The growth inhibitory effects of stattic and chemo agents including doxorubicin (DOX) and oxaliplatin (OXP) were evaluated using MTT assay in B16F10 and CT26 cell lines. Flow cytometry was applied to study cell apoptosis and calreticulin (CRT) surface exposure. The levels of high mobility group box 1 (HGMB1), heat shock protein70 (HSP70) and interleukin-12 (IL-12) were measured using ELISA. RESULTS: Treatment of B16F10 and CT26 cells with stattic in combination with DOX resulted in synergistic antitumor effects with combination index being 0.82 and 0.87, respectively. Interestingly, we found a higher level of ICD markers including CRT expression as well as HMGB1 and HSP70 secretion in the cells received combination therapy of stattic and DOX as compared with monotherapies. Moreover, exposure of dendritic cells (DCs) to conditioned media (CM) from cancer cells treated with stattic and/or DOX resulted in secretion of IL-12, which is an indicator of DCs maturation and induction of Th1 response. OXP and stattic monotherapy induced ICD in CT26 cells and stimulated IL-12 secretion by DCs; however, we did not observe a significant increase in the level of ICD in CT26 cells and IL-12 secretion by DCs when CT26 cells were treated with stattic and OXP combination as compared with monotherapy groups. CONCLUSION: These findings indicate that STAT3 inhibitory stattic can increase ICD induced by DOX. Graphical abstract.

Enhanced Production of Insulin-like Growth Factor I Protein in Escherichia coli by Optimization of Five Key Factors.

Human insulin-like growth factor I (hIGF-I) is a kind of growth factor with clinical significance in medicine. Up to now, E. coli expression system has been widely used as a host to produce rhIGF-1 with high yields. Batch cultures as non-continuous fermentations were carried out to overproduce rhIGF-I in E. coli. The major objective of this study is over- production of recombinant human insulin-like growth factor I (rhIGF-I) through a developed process by recruiting effective factors in order to achieve the most recombinant protein. In this study we investigated the effect of culture medium, induction temperature and amount of inducer on cell growth and IGF-1 production. Taguchi design of experiments (DOE) method was used as the statistical method. Analysis of experimental data showed that maximum production of rhIGF-I was occurred in 32y culture medium at 32 °C and 0.05 Mm IPTG. Under this condition, 0.694 g/L of rhIGF-I was produced as the inclusion bodies. Following optimization of these three factors, we have also optimized the amount of glucose and induction time in 5 liter top bench bioreactor. Full factorial design of experiment method was used for these two factors as the statistical method. 10 g/L and OD600=5 were selected as the optimum point of Glucose amount and induction time, respectively. Finally, we reached to a concentration of 1.26 g/L rhIGF-1 at optimum condition.