C-myc Gene Expression Research Articles

Activation of MEK‐ERK‐c‐MYC signaling pathway promotes splenic M2-like macrophage polarization to inhibit PHcH-liver cirrhosis

IntroductionPortal hypertension combined with hypersplenism (PHcH) is the main cause of hypocytosis and esophagogastric variceal hemorrhage in patients with liver cirrhosis. Activated macrophages that destroy excess blood cells are the main cause of hypersplenism, but the activating pathway is not very clear. This study aims to investigate the activation types of splenic macrophages and their activation mechanisms, to provide experimental evidence for the biological treatment of splenomegaly, and to find a strategy to improve liver fibrosis and inflammation by intervening in splenic immune cells. This study revealed the occurrence of M2-like polarization of macrophages and upregulation of c-Myc gene expression in the PH spleen.MethodsRNAseq, protein chip, western blot, and chip-seq were performed on macrophages and the in vitro MEK inhibitor rafametinib was used. Carbon tetrachloride and thioacetamide induced mouse cirrhosis models were separately constructed.Resultsc-Myc gene knockout in splenic macrophages reduced M2-like polarization and exacerbated liver fibrosis inflammation. c-Myc activated the MAPK signaling pathway and upregulated the expression of IL-4 and M2-like related genes in PH hypersplenism through the MEK-ERK-c-Myc axis. In addition, the c-Myc gene exerted anti-inflammatory effects by upregulating IL-4-mediated signal transduction to promote M2-like differentiation and anti-inflammatory cytokine secretion.ConclusionsActivation of MEK‐ERK‐c‐MYC signaling pathway promotes splenic M2-like macrophage polarization to inhibit PHcH-liver cirrhosis. Therefore, the induction of macrophage depolarization might represent a new therapeutic approach in the cure of PH hypersplenism, making c-Myc a potential candidate for macrophage polarization therapy.

Bile acid conjugated chitosan nanoparticles promote the proliferation and epithelial-mesenchymal transition of hepatocellular carcinoma by regulating the PI3K/Akt/mTOR pathway

Bile acids have been known to play significant roles at certain physiological levels in gastrointestinal metabolism. Yet, they are known to be carcinogenic and aid in tumor progression in most cases, although the roles remain uncertain. Hence, we tested the cytotoxic potential of cholic acid (CA) loaded chitosan nanoparticles (CNPs) on Hep3B cells. The physicochemical properties of the CNPs synthesized with CA load (CA-CNPs) were determined using standard techniques such as ultraviolet–visible spectrophotometry (UV–Vis), fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), dynamic light scattering (DLS) and transmission electron microscopy (TEM). The characteristic peak for chitosan nanoparticles were observed for plain CNPs (pCNPs) and CA-CNPs at around 300 nm as per UV–Vis analysis. FTIR analysis indicated the possible trapping of CA onto CNPs as certain peaks were retained and some peaks were shifted. XRD analysis determined that the peaks representing CA and pCNPs were collectively obtained in CA-CNPs. As per DLS analysis, the particle size, PDI and ζ-potential of the CA-CNPs were 259 nm, 0.284 and 30.4 mV. Further, the CA-CNPs were non-cytotoxic on Hep3B cells at the maximum tested concentration of 500 μg/mL. The viability at 500 μg/mL of CA-CNPs was two-fold higher than 500 μg/mL of pCNPs. Also, the pCNPs were not hemolytic and therefore could not have played a role in the increase of viability after treatment with CA-CNPs, which indicates that CA posed a major role in increased viability of Hep3B cells. As per quantitative PCR (qPCR), the upregulated gene expressions of PI3K, Akt, mTORC2, cMyc, Fibronectin, hVPS34, Slug and ZEB1 and the downregulated expression of the tumor suppressor PTEN indicates that PI3K/Akt/mTOR pathway mediated the induction of epithelial-to-mesenchymal transition (EMT) in response to CA-CNPs treatment on Hep3B cells.

LncRNA PVT1 silencing inhibits gastric cancer cells’ progression via enhancing chemosensitivity to paclitaxel

Gastric cancer (GC) is one of the leading causes of cancer-related deaths worldwide because of its high morbidity and the absence of effective therapies. Even though paclitaxel is a powerful anticancer chemotherapy drug, recent studies have indicated its ineffectiveness against GC cells. Long non-coding RNA (lncRNA) PVT1 has a high expression in GC cells and increases the progression of tumors via inducing drug resistance. In the present study, the effects of the siRNA-mediated lncRNA PVT1 gene silencing along with paclitaxel treatment on the rate of apoptosis, growth, and migration of AGS GC cells were investigated. AGS cells were cultured and then transfected with siRNA PVT1 using electroporation. The MTT test was used to examine the effect of treatments on the viability of cultured cells. Furthermore, the flow cytometry method was used to evaluate the impact of treatments on the cell cycle process and apoptosis induction in GC cells. Finally, the mRNA expression of target genes was assessed using the qRT-PCR method. The results showed that lncRNA PVT1 gene suppression, along with paclitaxel treatment, reduces the viability of cancer cells and significantly increases the apoptosis rate of cancer cells and the number of cells arrested in the G2/M phase compared to the control group. Based on the results of qRT-PCR, combined treatment significantly decreased the expression of MMP3, MMP9, MDR1, MRP1, Bcl-2, k-Ras, and c-Myc genes and increased the expression of the Bax gene compared to the control group. The results of our study showed that lncRNA PVT1 gene targeting, together with paclitaxel treatment, induces apoptosis, inhibits growth, alleviates drug resistance, and reduces the migratory capability of GC cells. Therefore, there is a need for further investigations to evaluate the feasibility and effectiveness of this approach in vivo in animal models.

Differential Regulation of Wingless-Wnt/c-Jun N-Terminal Kinase Crosstalk via Oxidative Eustress in Primary and Metastatic Colorectal Cancer Cells.

In the tumor microenvironment (TME), ROS production affects survival, progression, and therapy resistance in colorectal cancer (CRC). H2O2-mediated oxidative stress can modulate Wnt/β-catenin signaling and metabolic reprogramming of the TME. Currently, it is unclear how mild/moderate oxidative stress (eustress) modulates Wnt/β-catenin/APC and JNK signaling relationships in primary and metastatic CRC cells. In this study, we determined the effects of the H2O2 concentration inducing eustress on isogenic SW480 and SW620 cells, also in combination with JNK inhibition. We assessed cell viability, mitochondrial respiration, glycolysis, and Wnt/β-catenin/APC/JNK gene and protein expression. Primary CRC cells were more sensitive to H2O2 eustress combined with JNK inhibition, showing a reduction in viability compared to metastatic cells. JNK inhibition under eustress reduced both glycolytic and respiratory capacity in SW620 cells, indicating a greater capacity to adapt to TME. In primary CRC cells, H2O2 alone significantly increased APC, LEF1, LRP6, cMYC and IL8 gene expression, whereas in metastatic CRC cells, this effect occurred after JNK inhibition. In metastatic but not in primary tumor cells, eustress and inhibition of JNK reduced APC, β-catenin, and pJNK protein. The results showed differential cross-regulation of Wnt/JNK in primary and metastatic tumor cells under environmental eustress conditions. Further studies would be useful to validate these findings and explore their therapeutic potential.

Identification and validation of plasma AGRN as a novel diagnostic biomarker of hepatitis B Virus-related chronic hepatitis and liver fibrosis/cirrhosis.

The aim of this study was to find novel biomarkers and develop a non-invasive, effective diagnostic model for hepatitis B Virus-related chronic hepatitis and liver fibrosis/cirrhosis. Quantitative real-time polymerase chain reaction (qRT-PCR) was utilized to assess the expression of differentially expressed genes (AGRN, JAG1, CCL5, ID3, CCND1, and CAPN2) in peripheral blood mononuclear cells (PBMCs) from healthy subjects, chronic hepatitis B (CHB), and liver fibrosis/cirrhosis (LF/LC) patients. The molecular mechanisms underlying AGRN-regulated CHB were further explored and verified in LX2 cells, in which small interfering RNA (siRNA) was used to block AGRN gene expression. Finally, enzyme-linked Immunosorbent Assay (ELISA) was used to measure AGRN protein expression in 100 healthy volunteers, 100 CHB patients, and 100 LF/LC patients, and the efficacy of the diagnostic model was assessed by the Area Under the Curve (AUC). AGRN mRNA displayed a steady rise in the PBMCs of normal, CHB, and LF/LC patients. Besides, AGRN expression was markedly elevated in activated LX2 cells, whereas the expression of COL1 and α-SMA decreased when AGRN was inhibited using siRNA. In addition, downregulation of AGRN can reduce the gene expression of β-catenin and c-MYC while upregulating the expression of GSK-3β. Furthermore, PLT and AGRN were used to develop a non-invasive diagnostic model (PA). To identify CHB patients from healthy subjects, the AUC of the PA model was 0.951, with a sensitivity of 87.0% and a specificity of 91.0%. The AUC of the PA model was 0.922 with a sensitivity of 82.0% and a specificity of 90.0% when differentiating between LF/LC and CHB patients. The current study indicated that AGRN could be a potential plasma biomarker and the established PA model could improve the diagnostic accuracy for HBV-related liver diseases.

ZNF300 promotes proliferation and migration of hepatocellular carcinoma by upregulating c-MYC gene expression

BackgroundHepatocellular carcinoma (HCC) is the most common primary malignant tumor of the liver. Currently, the treatments of HCC are limited to surgical resection and liver transplantation, and there is no effective systemic therapy. ObjectivesTo investigate the regulatory mechanism of zinc finger protein 300 (ZNF300) in hepatocellular carcinoma (HCC). MethodsThe expressions of ZNF300 in HCC tissue samples and HCC cell lines (Hep3B, Huh7, SNU-387) were detected. ZNF300 overexpression vector (ZNF300) or shRNAZNF300 (shZNF300) was transfected into HCC cells to increase or inhibit ZNF300 expression. 5‐Ethynyl‐2′‐deoxyuridine assay (EdU), cell counting kit‐8 assay (CCK‐8) and transwell invasion assay were conducted to evaluate the proliferation, viability, migration, and invasion of HCC cells respectively. The expressions of tumor migration and invasion related proteins (matrix metallopeptidase 2 (MMP-2) and MMP-9), c-MYC, and MAPK/ERK signaling pathway related molecules (p-ERK1/2, ERK1/2, p-P38, P38) were determined by western blotting. Hep3B cells transfected with shZNF300 were subcutaneously injected into nude mice to perform tumor xenograft experiment. Tumor volume and weight were measured. ResultsZNF300 was upregulated in HCC tissues and cells. The expressions of MMP-2 and MMP-9 were increased in HCC cells after transfecting with ZNF300 but reduced in HCC cells transfected with shZNF300. Downregulation of ZNF300 inhibited HCC cell proliferation, migration, and invasion, while overexpression of ZNF300 showed the opposite effects. Moreover, the expressions of c-MYC and MAPK/ERK signaling pathway related molecules were increased after overexpression of ZNF300 but reduced after downregulating ZNF300. In tumor xenograft experiment, downregulation of ZNF300 reduced tumor volume and weight. ConclusionThe present study proved that downregulation of ZNF300 inhibited HCC growth by reducing c-MYC expression and MAPK/ERK signaling pathway.

Identification of placenta-specific protein 1 (PLAC-1) expression on human PC-3 cell line-derived prostate cancer stem cells compared to the tumor parental cells

Placenta-specific protein 1 (PLAC-1) is a gene primarily expressed in the placenta and the testis. Interestingly, it is also found to be expressed in many solid tumors, and it is involved in malignant cell features. However, no evidence has been reported regarding the relationship between PLAC-1 and cancer stem cells (CSCs). In the current research, we explored the expression of the PLAC-1 molecule in prostate cancer stem cells (PCSCs) derived from the human PC-3 cell line. The enrichment of PCSCs was achieved using a three-dimensional cell culture technique known as the sphere-formation assay. To confirm the identity of PCSCs, we examined the expression of genes associated with stemness and pluripotency, such as SOX2, OCT4, Nanog, C-Myc, and KLF-4, as well as stem cell differentiation molecules like CD44 and CD133. These evaluations were conducted in both the PCSCs and the original tumor cells (parental cells) using real-time PCR and flow cytometry. Subsequently, we assessed the expression of the PLAC-1 molecule in both enriched cells and parental tumor cells at the gene and protein levels using the same techniques. The tumor cells from the PC-3 cell line formed spheroids with CSC characteristics in a non-adherent medium. The expression of SOX2, OCT4, Nanog, and C-Myc genes (p < 0.01), and the molecules CD44 and CD133 (p < 0.05) were significantly elevated in PCSCs compared to the parental cells. The expression of the PLAC-1 molecule in PCSCs showed a significant increase compared to the parental cells at both gene (p < 0.01) and protein (p < 0.001) levels. In conclusion, it was indicated for the first time that PLAC-1 is up-regulated in PCSCs derived from human PC-3 cell line. This study may propose PLAC-1 as a potential target in targeted therapies, which should be confirmed through further studies.

Gene expression profiling of p53 and c-myc in HTLV-1 positive blood donors in Congo.

The HTLV-1 infection persists for life, remaining as asymptomatic viral reservoirs in most patients, ensuring the chain of transmission, but around 4% develop adult T-cell leukemia/lymphoma (ATLL). HTLV-1 is an oncogenic retrovirus that transforms CD4+ T lymphocytes and deregulates the lymphoproliferative pathways that contribute to the development of ATLL. To achieve cell transformation, most oncogenic retroviruses use proto-oncogene capture transduction, with proviral integration disrupting the expression of tumor suppressors or proto-oncogenes. We conducted this study on the prevalence of HTLV-1 infection in blood donors to expand the HTLV-1 database, assess the risk of transmission via blood products, as well as evaluate the risk of persistent infection or development of neoplastic diseases in HTLV-1 carriers. This is a cross-sectional study of blood donors of all categories. For this study, 265 blood donors were recruited at the Centre National de Transfusion Sanguine in Brazzaville. After testing for HTLV-1 antibodies by ELISA, proviral DNA was extracted from all ELISA-positive samples for detection by nested PCR, followed by RT qPCR using specific primers p53 and c-myc for gene expression. 20/265 were positive for anti-HTLV-1 antibody, 5 donors were positive for proviral DNA. The prevalence of HTLV-1 was 1.8%. All HTLV-1-positive donors were male (1.8%), with a positive correlation (p = 0.05); the 1.1% of positive donors were regular, with the majority aged between 31 and 45 years (1.5%), and concubine donors were the most frequent (1.1%). All samples showed normal expression of the p53 and c-myc genes. The prevalence, though low, remains a serious problem. No abnormal p53 or c-myc gene expression was detected in HTLV-1-positive donors, which could mean that none of the T lymphocytes in these donors had been transformed by HTLV-1.

Alleviation of doxorubicin adverse effects via loading into various drug-delivery systems: a comparative study.

Aim: Drug resistance is still a significant barrier to effective hepatocellular carcinoma therapy. Address the issue of doxorubicin resistance and inter-receptor crosstalk various doxorubicin formulations were investigated. Methods: Hepatocellular carcinoma was carried out using 3-methylechloroanthrene. Animals were then treated with doxorubicin, liposomal doxorubicin, titanium-loaded doxorubicin (TiO2-Dox), lactoferrin-doxorubicin and PEGylated doxorubicin. Biochemical and molecular analyses were assessed. Results: Results have declared a significant alternation of both sodium and potassium concentrations upon 3-methylechloroanthrene administration. Arginase-I and α-L-Fucodinase tumor biomarkers were significantly elevated. C-myc, Hprt-1 and EGFR gene expression were over-expressed. Treatment with the aforementioned treatment regimens significantly modulated all measured parameters. Conclusion: TiO2-Dox, doxorubicin-lactoferrin and PEGylated doxorubicin could be a promising regimen in hepatocellular carcinoma and overcoming the problem ofdrug resistance.

Restoration of miR-451a-5p/miR-34a-5p could suppress the proliferation and migration of human breast cancer cells through Wnt/β- catenin and ERK/P-ERK signaling pathways.

MicroRNAs (miRNAs) are a class of small non-coding RNAs with a length of 21-25 nucleotides and play an essential role in the regulation of cancer initiation, development and progression. Breast cancer (BC) is the most commonly detected malignancy in women and one of the leading causes of death worldwide. In this study, the effects of transfection of microRNA-451a-5p and miR-34a-5p (tumor suppressors), individually and in combination on apoptosis, proliferation and migration of breast cancer cells in vitro were investigated. For this study, malignant breast cancer cells (MDA-MB-231) were transfected with the miR-451a-5p and miR-34a-5p mimics. Subsequently cytotoxicity, apoptosis, proliferation, migration protein and gene expression of caspase-3, caspase-8, MMP9, ROCK, vimentin and c-Myc of the cancer cells were analyzed by MTT, flow cytometry, q-RT-PCR (expression level of caspase-3, caspase-8, MMP9, ROCK, vimentin and c-Myc genes), wound healing and Western blot assays. The results showed that miR-34a-5p and miR-451a-5p could additionally induce apoptosis and cell cycle arrest in the sub-G1phase, suppress proliferation and migration in breast cancer cells, and also decrease the expression of β- catenin and ERK/P-ERK proteins . The present data document that restoration of the tumor suppressor miR-451/miR-34 strongly induces programmed cell death in vitro and apparently inhibits cell proliferation and migration in human breast cancer cells. In summary, miR-451a and miR-34a play an important role in breast cancer cell proliferation and migration via the Wnt/β-catenin and ERK/P-ERK signaling pathways. Therefore, the simultaneous restoration of the presented tumor suppressor miRNAs can be proposed as a valuable and potential therapeutic strategy in the treatment of breast cancer. However, further studies should be useful.

Abstract 5982: Cannabinoid derived product is a potential novel therapeutic for papillary thyroid carcinoma

Abstract Papillary thyroid carcinoma (PTC) is the most common thyroid cancer that usually affects women ages 20-50, presenting as an asymptomatic neck mass. Total or partial thyroidectomy shows an excellent prognosis; however, investigation into non-invasive therapeutics with minimal adverse effects is ongoing. In addition to driver mutations such as BRAF, RAS, and RET-PTC, p53 mutations have also been identified. The loss of function of p53 is linked to cancer progression from PTC to poorly differentiated thyroid cancer and possibly anaplastic thyroid cancer. The disruption of the Wnt cell pathway is documented to be related to malignant cell transformation in thyroid cancer, leading to remarkable upregulation of c-myc, resulting in enhancement of tumor growth and drug resistance. Previous studies revealed that upregulation of BCL-2 occurs at later stages of tumorigenesis and is involved in chemotherapy resistance in some forms of cancer. In PTC, the downregulation of BCL-2 is linked to its favorable prognosis. Cannabinoid Derived Products (CDPs) have shown effectiveness in inhibiting different types of cancer, with research dating back to the 1970s. Ongoing in-vitro and in-vivo studies show that CDPs can effectively modulate tumor growth. However, the apoptotic properties of CDPs are largely dependent on the cancer type and drug dose/concentration. Our investigation focuses on CDPs and its potential anti-cancer effects by modulation of gene expression. We investigated its effects on gene expression of p53, c-Myc, and BCL-2 in K1 cell line which consists of PTC cells obtained from metastasis of well-differentiated PTC. K1 PTC cells (1 x106 cells/mL) were cultured with CDPs and incubated at 37°C under 5% CO2 for 24 and 48 hours. Cells were harvested, and cell viability was determined via trypan blue exclusion assay. Using qRT-PCR, we determined the effects of CDPs on the expression of TP53, c-Myc, and BCL-2. Results show that the CDPs decreased the viability (p&amp;lt; 0.001) of K1 PTC cells in a dose and time-dependent manner. Within 24 hours, the cannabinoid-containing product increased the gene expression of TP53 (p &amp;lt; 0.01) and decreased the gene expression of BCL-2 (p&amp;lt; 0.01) and c-Myc (p&amp;lt; 0.05) in K1 PTC cells. The results suggest that CDPs interact as a potential regulator in cancer with the upregulation of p53 and downregulation of BCL-2 and c-Myc. Further in-vitro and in-vivo studies are needed to understand the mechanism and therapeutic potential of cannabinoid-containing products in papillary thyroid cancer. Citation Format: Carolina Taico, Ibrahim Musa, Fariba Ardalani, Anish Maskey, Nan Yang, Joseph Breslin, Raj Tiwari, Jan Geliebter, Xiu Min Li. Cannabinoid derived product is a potential novel therapeutic for papillary thyroid carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5982.

Engineering mammalian cell growth dynamics for biomanufacturing

Precise control over mammalian cell growth dynamics poses a major challenge in biopharmaceutical manufacturing. Here, we present a multi-level cell engineering strategy for the tunable regulation of growth phases in mammalian cells. Initially, we engineered mammalian death phase by employing CRISPR/Cas9 to knockout pro-apoptotic proteins Bax and Bak, resulting in a substantial attenuation of apoptosis by improving cell viability and extending culture lifespan. The second phase introduced a growth acceleration system, akin to a “gas pedal”, based on an abscidic acid inducible system regulating cMYC gene expression, enabling rapid cell density increase and cell cycle control. The third phase focused on a stationary phase inducing system, comparable to a “brake pedal”. A tetracycline inducible genetic circuit based on BLIMP1 gene led to cell growth cessation and arrested cell cycle upon activation. Finally, we developed a dual controllable system, combining the “gas and brake pedals”, enabling for dynamic and precise orchestration of mammalian cell growth dynamics. This work exemplifies the application of synthetic biology tools and combinatorial cell engineering, offering a sophisticated framework for manipulating mammalian cell growth and providing a unique paradigm for reprogramming cell behaviour for enhancing biopharmaceutical manufacturing and further biomedical applications.

Distinguishing G-Quadruplexes Stabilizer and Chaperone for c-MYC Promoter G-Quadruplexes through Single-Molecule Manipulation.

G-quadruplex (G4) selective stabilizing ligands can regulate c-MYC gene expression, but the kinetic basis remains unclear. Determining the effects of ligands on c-MYC promoter G4s' folding/unfolding kinetics is challenging due to the polymorphic nature of G4s and the high energy barrier to unfold c-MYC promoter G4s. Here, we used single-molecule magnetic tweezers to manipulate a duplex hairpin containing a c-MYC promoter sequence to mimic the transiently denatured duplex during transcription. We measured the effects of six commonly used G4s binding ligands on the competition between quadruplex and duplex structures, as well as the folding/unfolding kinetics of G4s. Our results revealed two distinct roles for G4s selective stabilization: CX-5461 is mainly acting as c-MYC G4s stabilizer, reducing the unfolding rate (ku) of c-MYC G4s, whereas PDS and 360A also act as G4s chaperone, accelerating the folding rates (kf) of c-MYC G4s. qRT-PCR results obtained from CA46 and Raji cell lines demonstrated that G4s stabilizing ligands can downregulate c-MYC expression, while G4s stabilizer CX-5461 exhibited the strongest c-MYC gene suppression. These results shed light on the potential of manipulating G4s' folding/unfolding kinetics by ligands for precise regulation of promoter G4-associated biological activities.

SiRNA-mediated downregulation of BATF3 diminished proliferation and induced apoptosis through downregulating c-Myc expression in chronic myelogenous leukemia cells.

Despite considerable improvement in therapeutic approaches to chronic myeloid leukemia (CML) treatment, this malignancy is considered incurable due to resistance. However, investigating the molecular mechanism of CML may give rise to the development of extremely efficient targeted therapies that improve the prognosis of patients. Basic leucine zipper transcription factor ATF-like3 (BATF3), as transcription factor, is considered a key regulator of cellular activities and its function has been evaluated in tumor development and growth in several cancer types. This study aimed to evaluate the potential of the cellular impact of siRNA-mediated downregulation of BATF3 on CML cancer cells through cell proliferation, induction of apoptosis, and cell cycle distribution. The transfection of BATF3 siRNA to K562 CML cells was performed by electroporation device. To measure cellular viability and apoptosis, MTT assay and Annexin V/PI staining were carried out, respectively. Also, cell cycle assay and flow cytometry instrument were applied to assess cell cycle distribution of K562 cells. For more validation, mRNA expression of correlated genes was relatively evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). The data indicated that siRNA-mediated BATF3 inactivating severely promoted the cell apoptosis. Also, the targeted therapy led to high expression of Caspase-3 gene and Bax/Bcl-2 ratio. Silenced BATF3 also induced cell cycle arrest in phase sub-G1 compared to control. Finally, a noticeable decrement was obtained in c-Myc gene expression through suppression of BATF3 in CML cells. The findings of this research illustrated the suppression of BATF3 as an effective targeted therapy strategy for CML.

Capsaicin: A chili pepper bioactive phytocompound with a potential role in suppressing cancer development and progression.

Cancer profoundly influences morbidity and fatality rates worldwide. Patients often have dismal prognoses despite recent improvements in cancer therapy regimens. However, potent biomolecules derived from natural sources, including medicinal and dietary plants, contain biological and pharmacological properties to prevent and treat various human malignancies. Capsaicin is a bioactive phytocompound present in red hot chili peppers. Capsaicin has demonstrated many biological effects, including antioxidant, anti-inflammatory, antimicrobial, and anticarcinogenic capabilities. This review highlights the cellular and molecular pathways through which capsaicin exhibits antineoplastic activities. Our work also depicts the synergistic anticancer properties of capsaicin in conjunction with other natural bioactive components and approved anticancer drugs. Capsaicin inhibits proliferation in various cancerous cells, and its antineoplastic actions in numerous in vitro and in vivo carcinoma models impact oncogenesis, tumor-promoting and suppressor genes, and associated signaling pathways. Capsaicin alone or combined with other phytocompounds or approved antineoplastic drugs triggers cell cycle progression arrest, generating reactive oxygen species and disrupting mitochondrial membrane integrity, ultimately stimulating caspases and promoting death. Furthermore, capsaicin alone or in combination can promote apoptosis in carcinoma cells by enhancing the p53 and c-Myc gene expressions. In conclusion, capsaicin alone or in combination can have enormous potential for cancer prevention and intervention, but further high-quality studies are needed to firmly establish the clinical efficacy of this phytocompound.

Inhibition of Myeloma Cell Function by Cannabinoid-Enriched Product Associated With Regulation of Telomere and TP53.

Multiple myeloma is a hematological cancer caused by the uncontrolled proliferation of abnormal plasma cells in the bone marrow, leading to excessive immunoglobulin production. Our study aimed to examine the anticancer properties of BRF1A, a cannabinoid (CBD)-enriched product, on 2 myeloma cell lines: U266 and ARH-7. We treated U266 and ARH-77 myeloma cells with varying doses of BRF1A and measured the production of IgE and IgG antibodies using ELISA. Cell viability was assessed using trypan blue and CCK-8 assays. We measured the expression of genes related to the production of IgE and IgG antibodies, IgEH, and IgGH. We determined its effect on the expression of telomerase and its phosphorylated form as an indicator of telomere stabilization. Furthermore, we determined its effect on other cancer-related targets such as NF-ĸB, c-Myc, and TP53 in U266 cells using reverse transcription polymerase chain reaction (RT-PCR) and western blotting. BRF1A reduced myeloma cell IgE and IgG production in a time and dose-dependent manner. It also suppressed the expression of p-IκBα, p-NFκB (p65), and total NFκB protein, as well as XBP1u and XBP1s. It increased the gene and protein expression of telomere and hTERT and significantly increased cancer suppressor TP53 gene and p53 protein expression. Additionally, BRF1A decreased the c-Myc gene and protein expression. Our study has shown that a CBD-enriched product can reduce the growth of myeloma cells by suppressing the critical functions of IgE- and IgG-producing cells. This study could help bridge the gap in understanding how cannabinoid-containing products affect cancer, aging, telomere, and cancer-suppressor gene activity.

Investigation of iso-propylchaetominine anticancer activity on apoptosis, cell cycle and Wnt signaling pathway in different cancer models

Dysregulation of the Wnt signaling pathway contributes to the development of many cancer types. Natural compounds produced with biotechnological systems have been the focus of research for being a new drug candidate both with unlimited resources and cost-effective production.In this study, it was aimed to reveal the effects of isopropylchaetominine on cytotoxic, cytostatic, apoptotic and Wnt signaling pathways in brain, pancreatic and prostate cancer.The IC50 values of isopropylchaetominine in U-87 MG, PANC1, PC3 and LNCaP cells were calculated as 91.94 μM, 41.68 μM, 54.54 μM and 7.86 μM in 72nd h, respectively. The metabolite arrests the cell cycle in G0/G1 phase in each cancer cells. Iso-propylchaetominine induced a 4.3-fold and 1.9-fold increase in apoptosis in PC3 and PANC1 cells, respectively. The toxicity of isopropylchaetominine in healthy fibroblast cells was assessed using the annexin V method, and no significant apoptotic activity was observed between the groups treated with the active substance and untreated. In U-87 MG, PANC1, PC3, and LNCaP cells under treatment with isopropylchaetominin, the expression levels of DKK3, TLE1, AES, DKK1, FRZB, DAB2, AXIN1/2, PPARD, SFRP4, APC and SOX17 tumor suppressor genes increased significantly. Decreases in expression of Wnt1, Wnt2, Wnt3, Wnt4, Wnt5, Wnt6, Wnt10, Wnt11, FRZ2, FRZ3, FRZ7, TCF7L1, BCL9, PYGO, CCND2, c-MYC, WISP1 and CTNNB1 oncogenic genes were detected.All these result shows that isopropylchaetominine can present promising new treatment strategy in different cancer types.

Novel Protac-Based EZH2 Degraders Effectively Target EZH2-Driven Lymphoid Malignancies By Abolishing Both Methylation-Dependent and -Independent Functions

Across various cancers, overexpression or mutation of the epigenetic silencer EZH2 correlates with invasive cancer growth, resistance and poor survival outcomes. While the tumorigenic role of EZH2 is largely attributed to the H3K27me3-mediated repression of tumor suppressor genes, reports have also established a methylation-independent role for EZH2 in the transcriptional activation of oncogenes, particularly in aggressive cancers like Natural Killer T-cell Lymphoma (NKTL). Here, traditional EZH2 inhibitors have been ineffective. We hypothesized that targeted depletion of the EZH2 protein will more effectively disrupt oncogenic signals arising from enzyme-independent activities of EZH2. Thus, we embarked on the development of EZH2 Proteolysis Targeting Chimeras (PROTACs) to trigger targeted ubiquitination and proteasome-mediated degradation of EZH2. No clinical EZH2 PROTAC has been reported yet. Our initial efforts focused on the iterative synthesis of a series of PROTAC candidates considering factors such as permeability, solubility and stable ternary complex formation. We developed a library of 53 compounds with various permutations of EZH2- and E3 ligase-binding ligands as well as linker lengths. Through extensive screening and testing of the EZH2 degradation activity via the Nano-Glo HiBiT lytic detection system and immunoblotting, we identified three lead Protac candidates. Compared to the clinical EZH2 inhibitor Tazemetostat (Taz), EZH2 PROTACs, C2911 and C1311 demonstrated higher cytotoxic activity in diffuse large B-cell lymphoma (DLBCL), a cancer in which Taz was evaluated during clinical trial. Importantly, C2911 and C1311 also successfully triggered EZH2 degradation in a dose-dependent manner thereby qualifying for further evaluation in NKTL. Previous work has highlighted a critical role for JAK3-mediated phosphorylation of EZH2 in promoting NKTL proliferation. Treatment of NKTL with C2911 and C1311 leads to a dose and time-dependent EZH2 degradation, loss of cell viability and increased apoptosis. These effects were absent with Taz and Pomalidomide treatments. The concomitant loss of H3K27me3 levels as well as PRC2 complex subunits, SUZ12 and EED suggest effective degradation of the EZH2 protein. We also observed that induction of NKTL cell death required a longer treatment period as compared to DLBCL. This may be potentially ascribed to the longer half-life of the EZH2 protein in NKTL as determined through cycloheximide treatment. In order to validate the mechanism of action of the EZH2 PROTACs, cells were either pretreated with pomalidomide or siRNA targeting cereblon. Indeed, this mitigated PROTAC-induced degradation of the EZH2 protein and rescued cancer cell death. Similarly, inhibiting the activity of the 26S proteasome through bortezomib also prevented EZH2 degradation. Characterization of EZH2 PROTAC-induced protein expression changes through immunoblotting highlighted that there was specific loss of H3K27me3 and H3K27me2 but not H3K27me1. Of note, we detected a concomitant decrease of Jak3 protein expression and STAT5 phosphorylation levels, more pronounced than PRC2 complex subunits. This suggests that the PROTAC may act by disrupting the non-canonical binding of EZH2 to Jak3 in NKTL. Furthermore, gene expression analyses revealed that EZH2 PROTAC treatment more effectively reactivated canonical EZH2 target genes such as MYT1 and ANPEP as compared to Taz. As the JAK-STAT-MYC axis is dysregulated in NK leukemia, we also evaluated the gene expression of c-Myc and c-Myc target gene, E2F1. EZH2 PROTAC treatment reduces the transcription levels of these oncogenes corroborating with loss of c-Myc protein expression. Studying the pharmacokinetics of the lead PROTACs in a mouse model revealed good plasma stability, half-life (&amp;gt; 4 h), and low clearance. Collectively, our data supports a critical role for EZH2 PROTACs in abolishing both the enzyme-dependent and independent activities of EZH2 through protein degradation, resulting in enhanced anti-cancer activity against DLBCL and NKTL. This may potentially benefit other EZH2-driven cancers. Further evaluation of the EZH2 PROTACs for clinical application is in progress. EZH2 PROTACs will next be tested in vivo. An in-depth investigation of the mechanisms stabilizing EZH2 protein levels and underlying PROTAC induced-cell death will be made to identify potential mechanisms of resistance.

Cooperatively inhibition effect of miR-143-5p and miR-145-5p in tumorigenesis of glioblastoma cells through modulating AKT signaling pathway

Introduction: As the most common aggressive primary brain tumor, glioblastoma is inevitably a recurrent malignancy whose patients’ prognosis is poor. miR-143 and miR-145, as tumor suppressor miRNAs, are downregulated through tumorigenesis of multiple human cancers, including glioblastoma. These two miRNAs regulate numerous cellular processes, such as proliferation and migration. This research was intended to explore the simultaneous replacement effect of miR-143, and miR-145 on in vitro tumorgenicity of U87 glioblastoma cells. Methods: U87 cells were cultured, and transfected with miR-143-5p and miR-145-5p. Afterward, the changes in cell viability, and apoptosis induction were determined by MTT assay and Annexin V/PI staining. The accumulation of cells at the cell cycle phases was assessed using the flow cytometry. Wound healing and colony formation assays were performed to study cell migration. qRT-PCR and western blot techniques were utilized to quantify gene expression levels. Results: Our results showed that miR-143-5p and 145-5p exogenous upregulation cooperatively diminished cell viability, and enhanced U-87 cell apoptosis by modulating Caspase-3/8/9, Bax, and Bcl-2 protein expression. The combination therapy increased accumulation of cells at the sub-G1 phase by modulating CDK1, Cyclin D1, and P53 protein expression. miR-143/145-5p significantly decreased cell migration, and reduced colony formation ability by the downregulation of c-Myc and CD44 gene expression. Furthermore, the results showed the combination therapy of these miRNAs could remarkably downregulate phosphorylated-AKT expression levels. Conclusion: In conclusion, miR-143 and miR-145 were indicated to show cooperative anti- cancer effects on glioblastoma cells via modulating AKT signaling as a new therapeutic approach.

Targeting RAS-ERK pathway alterations with MEK inhibitors to improve chemosensitivity in high grade serous ovarian cancers

ObjectiveAssess if MEK inhibitor blockade of RAS-ERK pathway adaptive response in high grade serous ovarian cancers (HGSOC) improves platinum sensitivity. MethodsThree HGSOC cell lines and three patient derived organoid (PDOs) samples from ascites of platinum resistant HGSOC patients were collected. Cell lines and PDOs were exposed to carboplatin and MEK inhibitors cobimetinib or trametinib. Cytotoxic effects of MEK inhibitors alone or combined with carboplatin were established. Western blots demonstrated RAS-ERK pathway blockage after MEK inhibitor treatment. RNA sequencing assessed gene expression after MEK inhibitor treatment. Cell line NF1 gene knockdown was performed with corresponding chemosensitivity levels. ResultsHigh carboplatin IC50 levels indicated platinum resistance in cell lines and PDOs. Cobimetinib induced cytotoxicity in cell lines and PDOs, while trametinib was less effective. Western blot confirmed MEK-ERK pathway blockage at minimal concentrations of MEK inhibitors in cell lines and PDOs. Phosphorylated-ERK levels of untreated cells indicated higher levels of RAS-ERK pathway activation in OVSAHO and OVCAR7 compared to OVCAR3. OVSAHO harbors a NF1 mutation and had highest levels of RAS-ERK activation. Cotreatment with carboplatin and MEK inhibitors showed varying synergistic cytotoxic effects at different combinations. Synergistic effect was most prominent in the OVSAHO carboplatin and cobimetinib combination. RNA sequencing identified downregulation of c-MYC and FOXM1 gene expression after MEK inhibitor treatment. NF1 gene knockdown showed an acquired increased IC50 compared to parental cells. ConclusionMEK inhibitors block RAS-ERK pathways in platinum resistant HGSOC cells and PDOs. MEK inhibitors with carboplatin have select synergistic effects which may indicate a strategy to improve platinum sensitivity.