Expression Levels Of Bcl-xL Research Articles

The molecular impact of miR-326 in acute lymphoblastic leukemia and its cross talk with P53.

MiR-326 downregulation is strongly associated with multidrug resistance (MDR) and has been identified as an adverse prognostic biomarker for pediatric acute lymphoblastic leukemia (pALL). The choice to study miR-326 as a tumor suppressor in cancer biology, particularly its regulation of apoptosis, drug resistance, and stemness, stems from its strong association with MDR and potential as a therapeutic target in pALL. The current study aimed to investigate, for the first time, the molecular mechanisms underlying the role of miR-326 in ALL, using Gene Ontology annotation network and multilayer network analysis. Our findings revealed that miR-326 exhibits a multifunctional anti-tumor behavior, affecting various aspects of drug resistance, stemness, and apoptosis in cancer, particularly in the context of ALL. Quantitative real-time PCR demonstrated downregulation of the ABC transporter mRNAs ABCC1 and ABCB1 but not ABCA3 in B-ALL cells transfected with miR-326 mimic, as confirmed by bioinformatic data. Western blot analysis showed a possible cross talk between miR-326 and P53 through the upregulation of Mdm2 and P53 proteins. The heightened functional activity of P53 was subsequently validated through the observed augmentation in levels of P21 and CCND1, alongside the evident disruption in the expression levels of Bcl-2, Bcl-xl, and Bax genes. Subsequently, the ceRNA network between miR-326 and LncRNAs was exhibited and the impact of exogenous miR-326 on the expression levels of its molecular sponges, H19 and SNHG1 was examined using RT-qPCR. Future studies will explore the potential impact of miR-326 on its targets, and how this may influence the development of novel therapeutic strategies for ALL.

Effects of Seaweed Polysaccharide on the Growth and Physiological Health of Largemouth Bass, Micropterus salmoides.

A seven-week trial was designed to evaluate the effects of dietary seaweed polysaccharide (SP) supplementation on the growth performance and physiological health of largemouth bass. The results reveal that the 0.05SP group showed the best growth performance. The mRNA expression levels of tor, 4ebp1, and igf1 genes were remarkably down-regulated in the 0.15SP and 0.2SP groups compared to the control group. The CAT activities were significantly increased in the 0.05SP and 0.1SP groups, and the GSH-Px activity was increased in the 0.15SP group. The expression of the immune response-related gene nfκb was significantly down-regulated in the 0.1SP group, and those of tnfα and il-8 were at the maximum in the control group. Moreover, the expression of il-10 in the 0.15SP and 0.2SP groups was significantly down-regulated. Furthermore, endoplasmic reticulum stress (ERS)-related expression of atf6 was the highest in the control group. Furthermore, the chopα and bax expression levels in the 0.15SP and 0.2SP groups were significantly down-regulated compared with other groups. In addition, the highest expression level of bcl-xl was observed in the 0.15SP group. Finally, the quadratic regression analysis of antioxidant, immune, and ERS core parameters (CAT, nf-κb, and bcl-xl) determined 0.06-0.11% to be the optimal SP supplemental level in largemouth bass diets.

GdX inhibits the occurrence and progression of breast cancer by negatively modulating the activity of STAT3

ABSTRACT Aim To elucidate the biological functionality and regulatory mechanisms of GdX in breast cancer (BC). Methods The examination of GdX expression in human BC tissues and cell lines was conducted through immunohistochemical (IHC) and Western blot. Cell proliferation capacity was assessed via the CCK-8 and colony formation assay, while cell migration was determined through the wound healing assay. The expression levels of BCL-XL, Cyclin D1, and C-myc gene were quantified using RT-qPCR and Western blot. In vivo tumor growth was evaluated in nude mice xenografted with MDA-MB-231 cells overexpressing GdX, and a mouse model with GdX-deficient BC was established to observe the impact of GdX on BC formation and metastasis. Dual-luciferase reporter assay and immunofluorescence were employed to confirm the interaction between GdX and STAT3. Western blot was employed to validate the influence of GdX overexpression on the phosphorylation process of STAT3. Results GdX exhibited low expression in the cancer tissues of BC patients and cell lines. MDA-MB-231 and MCF-7 cells overexpressing GdX displayed a notable reduction in proliferation and diminished migratory capabilities, accompanied by downregulated mRNA and protein expression of BCL-XL, Cyclin D1, and C-myc. In the xenograft mouse model, heightened GdX expression correlated with a decelerated in vivo tumor growth. Furthermore, in mice deteleing GdX, both the quantity and weight of tumors increased, along with evident pulmonary metastasis. Mechanistically, STAT3 emerged as a downstream target gene of GdX. Conclusions GdX exerts its inhibitory effects on the initiation and progression of BC by negatively modulating the phosphorylation of STAT3.

Clinical Analysis of Multidrug Resistance-Related Protein Expression in Gastric Cancer Cells

Objective: This study aims to clinically analyze the expression of multidrug resistance-related proteins in gastric cancer cells. Methods: In this study, archival paraffin blocks from 50 surgical specimens of gastric cancer collected in our hospital between 2021 and 2022 were included. Each specimen included tumor lesion tissue and normal gastric mucosa tissue located more than 5 cm away from the cancer. Immunohistochemistry was performed to detect P-glycoprotein (P-gp), glutathione S-transferase pi (GST-π), and B-cell lymphoma-extra-large (Bcl-xL) expression, and the relevant data were collected and analyzed. Results: The positive expression rates and high expression levels of P-gp, GST-π, and Bcl-xL in gastric cancer tissues were significantly higher than in normal gastric mucosa tissues (p < 0.05). The expression of multidrug resistance proteins (P-gp, GST-π, Bcl-xL) in gastric cancer was not associated with patient clinical characteristics (tumor clinical stage, depth of invasion, and lymph node metastasis) (p > 0.05); Bcl-xL expression level was positively correlated with tumor differentiation degree (p < 0.05). Conclusion: The results of this study reveal the expression patterns of multidrug resistance-related proteins in gastric cancer tissues and shed light on the relationship between the expression of these proteins and multidrug resistance in gastric cancer.

The therapeutic potential and molecular mechanism of Alpha-pinene, Gamma-terpinene, and P-cymene against melanoma cells

The purpose of this study is to investigate the potential anticarcinogenic effects of three phytochemicals, namely Alpha-pinene (AP), Gamma-terpinene (GT), and P-cymene (PC), on melanoma cells (A2058 cell line). Additionally, the study aims to explore the synergistic activities of these phytochemicals with Dacarbazine, a chemotherapy drug. To understand the molecular mechanism involved in apoptosis induction in the A-2058 cell line, it was used AO/EB staining for apoptosis detection and cell cycle analysis, monitored through flow cytometry. It also determined the mRNA expression levels of different apoptosis-regulatory genes, including p53, Bax, NF-kB, Bcl-2, Bcl-xl, and caspase-3. The antitumor activities of these phytochemicals and their combinations were investigated in a subcutaneous mouse tumor model. The tumor diameter was 21.4 ± 1.1 mm in the Dacarbazine treatment group and 42.4 ± 3.1 mm in the control group. The antitumoral activities of AP and PC in the tumor model were similar to those of Dacarbazine. On the other hand, GT exhibited remarkable antitumoral activity, with a 1.75-fold reduction in tumor diameter compared to the Dacarbazine group. When different combinations of phytochemicals and Dacarbazine were used, the GT plus Dacarbazine treatment group was found to have a 3.5-fold reduction in tumor diameter compared to the Dacarbazine group. The tumor diameters in the Dacarbazine, AP plus GT, GT plus Dacarbazine, and AP plus Dacarbazine treatment groups were 21.4 ± 1.1, 7.6 ± 2.2, 8.6 ± 0.5, and 6.2 ± 1.9 mm, respectively.

Artesunate promotes cervical cancer cell apoptosis by regulating Bcl2 family molecules and reducing the mitochondrial membrane potential.

Artesunate (ART), an antimalarial drug, has a broad spectrum of antitumour effects in cancer types such as esophageal and gastric cancer. However, evidence demonstrating the role of ART in cervical cancer cells is limited. In the present study, the inhibitory effect of ART on the growth of cervical cancer cells through the modulation of the cell cycle and apoptosis was investigated. The growth-inhibitory effect of ART on a cervical cancer cell line (SiHa) was detected using a Cell Counting Kit-8 assay after treatment with ART for 24 h, after which the half-maximal inhibitory concentration (IC50) was calculated. Using flow cytometry assays, apoptosis, the cell cycle, the levels of reactive oxygen species (ROS) and calcium (Ca2+) ions, as well as the mitochondrial membrane potential were evaluated in SiHa cells following treatment with ART for 24 and 48 h. The mRNA expression levels of Bcl2, Bcl-xl, (myeloid cell leukaemia 1) Mcl-1, Bcl2-like protein 11 (BIM), (Bcl2-related ovarian killer protein) Bok, Bax and (Bcl2 homologous antagonist/killer) Bak in SiHa cells were detected using reverse transcription-quantitative PCR. ART inhibited the growth of SiHa cells in a dose-dependent manner. The IC50 of ART in SiHa cells was 26.32 µg/ml. According to the IC50 value, 15, 30 and 100 µg/ml ART were selected for further experiments, and normal saline (0 µg/ml ART) was used as the control group. The results indicated that treatment with 15, 30 and 100 µg/ml ART for 24 and 48 h induced apoptosis, increased the levels of ROS, the levels of Ca2+ and the mRNA expression levels of BIM, Bok, Bax and Bak, but decreased the cell proliferation indices, the mitochondrial membrane potential and the mRNA expression levels of Bcl2, Bcl-xl and Mcl-1 in a dose- and time-dependent manner. In conclusion, ART inhibited the growth of SiHa cells and induced apoptosis via a mechanism associated with the regulation of Bcl2 family member expression, which was associated with the increase of the levels of ROS and Ca2+ and the reduction of the mitochondrial membrane potential.

Curcumin Combined with Thalidomide Inhibits Proliferation of KG-1 Cells and Its Related Mechanisms

To investigate the effects of curcumin combined with thalidomide on the proliferation and apoptosis of acute myeloid leukemia KG-1 cells, and its correlation with B-cell lymphoma-xL (Bcl-xL), signal transducer and activator of transcription 3 (STAT3). MTT assay was used to detect the proliferation of KG-1 cells and screen the optimal combined concentration of curcumin and thalidomide. The effects of curcumin, thalidomide and their combination on the proliferation and apoptosis of KG-1 cells were analyzed by MTT method and flow cytometry, respectively. The mRNA expression levels of STAT3 and Bcl-xL in single-drug group, two-drug combination group and control group (untreated cells) were detected by real-time quantitative PCR. Both curcumin and thalidomide inhibited the proliferation of KG-1 cells in a concentration-dependent manner in the range of 20-100 μmol/L (r =0.657, r =0.681). The IC50 value of curcumin and thalidomide at 48 h was (42.07±0.50) μmol/L and (57.01±2.39) μmol/L, respectively. The cell proliferation inhibition rate of curcumin (40 μmol/L) + thalidomide (60 μmol/L) was (86.67±1.53)%, which was significantly higher than (51.67±1.15)% of curcumin (40 μmol/L) and (55.33±1.53)% of thalidomide (60 μmol/L) (both P < 0.05). Treated with curcumin and thalidomide alone or in combination, the apoptosis rate of KT-1 cells was (18.67±2.08)%, (21.33±2.52)%, and (46.67±1.53)%, respectively, which was significantly higher than (0.72±0.03)% of control group (all P < 0.05). The cell apoptosis rate of two-drug combination group was significantly higher than that of single-drug group (both P < 0.05). Compared with the control group, the mRNA expressions of STAT3 and Bcl-xL in single-drug group, two-drug combination group were significantly decreased (both P < 0.05). Compared with single-drug group, the mRNA expressions of STAT3 and Bcl-xL in two-drug combination group were also significantly decreased (both P < 0.05). Curcumin combined with thalidomide can synergistically down-regulate the expression of STAT3 and Bcl-xL, inhibit the proliferation of KG-1 cells, and induce apoptosis.

Abstract 7579: Dual inhibition of enhancer of zeste homolog 1 2 induces pyroptosis, modulates oncogenic signaling pathways &amp; sensitizes hepatocellular carcinoma to valemetostat &amp; docetaxel treatments

Abstract HCC is a global health concern, with chemotherapy resistance and patient recurrence posing significant challenges to liver cancer treatment. EZH2 is a crucial part of the polycomb repressive complex 2 (PRC2). It, along with its homolog EZH1, is a focus for cancer therapeutic targets. Dysregulation of EZH1/2 can cause cancer development due to significant changes in geneexpression. The current study aims to investigate the interaction between EZH1/2 and oncogenic signals responsible for HCC progression and pyroptosis induction. Two cell lines, HepG2 and SK-Hep1, were subjected to two FDA-approved drugs, Valemetostat (Val) and Docetaxel (Doc), to determine the impact of EZH1/2 on tumorigenicity and drug resistance. The WST-1 assay was utilized to gauge cell proliferation. The results indisputablydemonstrated a synergistic effect of the drugs after 48 h of treatment. The inhibition of viability was attributed to apoptosis induction in both tested cell lines, and the maximum effect was observed in the combined treatment as compared to either the control or each drug alone. Furthermore, our datashowed activation of caspase-3, upregulation of BAX, indicating apoptotic induction, and downregulation of Bcl-2, Bcl-XL, AKT, S6k, oncogenic β-catenin, and Stat3 protein expression levels, suggesting a modulation of oncogenic signaling pathways at the protein level. After 24 h of Val/Doc treatment, pyroptosis was observed, characterized by cell swelling and bubbles on themembrane and a significant release of lactate dehydrogenase, indicating pyroptotic cell cytotoxicity. The antitumor effects and pyroptosis were enhanced by using a dual inhibitor, UNC1999, for EZH1/2, resulting in a marked increase in the sensitivity of tested cells to the Val/Doc treatment invitro. The present study unequivocally demonstrated that dual inhibition of EZH1/2 acts as a potent sensitizer of HCC cells to Val/Doc treatment, which might be a promising therapeutic approach for inducing cellular pyroptosis in HCC cell lines. Citation Format: Asmaa Elkhder Harira, Adham Maher, Ahmed Sultan. Dual inhibition of enhancer of zeste homolog 1 2 induces pyroptosis, modulates oncogenic signaling pathways &amp; sensitizes hepatocellular carcinoma to valemetostat &amp; docetaxel treatments [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 7579.

Abstract 7589: The novel complex delivery system of C-phycocyanin-loaded klebsiella pneumoniabacterial ghost induces apoptosis and sensitizes non-small cell lung cancer cell lines to doxorubicin

Abstract Lung cancer is a significant cause of cancer-related deaths worldwide, with a high incidence rate, poor prognosis, aggressiveness, and a lack of effective treatment options. The present study has brought together two FDA-approved drugs with antineoplastic properties to create a complex that promises to revolutionize the treatment of Non-Small Cell Lung Cancer (NSCLC). By combining C-Phycocyanin extract (PCE), a pigment-protein complex synthesized by blue-green microalgae platensis, with the immunostimulatory doxorubicin (DOX) loaded Bacterial Ghosts (BGs), the present study aims to achieve enhanced dual-chemotherapy and remarkable inhibition of DOX-chemoresistance. The BG delivery system uses purified bacterial cell evacuation and safe confirmed Klebsiella pneumoniae BGs (KPBG) for safe and effective delivery, overcoming issues with resistance and poor delivery. The complex delivery system of KPBG-PCE/DOX was tested on two NSCLC cell lines, wild-P53, A549, and mutant-P53, NCI/H358, respectively. Our data showed that the loaded combined treatment significantly induced morphological changes with signs of apoptosis induction in the tested cell lines compared with the control. The WST-1 assay was used to evaluate cell proliferation. Data indicates that PCE and DOX have a synergistic effect at low concentrations when loaded on KPBGs for a constant count of BGs CFU/ml after 48 h of treatment. The viability inhibition and apoptosis induction of A549 and NCI/H358 cells were amplified in the combined treatment of 48 h compared to the control or either each drug alone. At the protein levels, our data showed activation of caspase-3, upregulation of BAX, and downregulation of Bcl-2, Bcl-XL, m-TOR, S6k, oncogenic β-catenin, and cyclin-D1 protein expression levels after treatment with (KPBG-PCE/DOX) for 48 h, indicating apoptotic induction and modulating oncogenic signaling pathways. In conclusion, the current study presents a promising outcome of a synergistic delivery system of KPBG-PCE/DOX that can inhibit lung cancer cell line proliferation, induce apoptosis, and increase cell sensitivity to DOX. The KPBG delivery system offers an appealing approach for a successful combination of targeted therapy for lung cancer. Citation Format: Basma Adel Elsayed Abdelaziz, Hoda Eid Mahmoud, Omayma Mohamed Sadek, Ahmed Samir Sultan. The novel complex delivery system of C-phycocyanin-loaded klebsiella pneumoniabacterial ghost induces apoptosis and sensitizes non-small cell lung cancer cell lines to doxorubicin [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 7589.

Investigation of apoptotic effects of Cucurbitacin D, I, and E mediated by Bax/Bcl-xL, caspase-3/9, and oxidative stress modulators in HepG2 cell line.

Cucurbitacins, natural compounds highly abundant in the Cucurbitaceae plant family, are characterized by their anticancer, anti-inflammatory, and hepatoprotective properties. These compounds have potential as therapeutic agents in the treatment of liver cancer. This study investigated the association of cucurbitacin D, I, and E (CuD, CuI, and CuE) with the caspase cascade, Bcl-2 family, and oxidative stress modulators in the HepG2 cell line. We evaluated the antiproliferative effects of CuD, CuI, and CuE using the MTT assay. We analyzed Annexin V/PI double staining, cell cycle, mitochondrial membrane potential, and wound healing assays at different doses of the three compounds. To examine the modulation of the caspase cascade, we determined the protein and gene expression levels of Bax, Bcl-xL, caspase-3, and caspase-9. We evaluated the total antioxidant status (TAS), total oxidant status(TOS), superoxide dismutase (SOD), glutathione (GSH), Total, and Native Thiol levels to measure cellular redox status. CuD, CuI, and CuE suppressed the proliferation of HepG2 cells in a dose-dependent manner. The cucurbitacins induced apoptosis by increasing caspase-3, caspase-9, and Bax activity, inhibiting Bcl-xL activation, causing loss of ΔΨm, and suppressing cell migration. Furthermore, cucurbitacins modulated oxidative stress by increasing TOS levels and decreasing SOD, GSH, TAS, and total and native Thiol levels. Our findings suggest that CuD, CuI, and CuE exert apoptotic effects on the hepatocellular carcinoma cell line by regulating Bax/Bcl-xL, caspase-3/9 signaling, and causing intracellular ROS increase in HepG2 cells.

Protective benefits of ethyl alcohol extract of Piper betel L. to prevent colon carcinogenesis

Piper betel L. is a traditional nutritional and medicinal food, known for its rich phytochemical components. In the current study, the role of the ethyl alcohol extract of betel leaf (Piper betel) against human colorectal carcinoma (HT29 cells), was investigated. The extract consists of phenolic compounds and flavonoids that are potent antioxidants and antimicrobial agents. Exposure of human colon carcinoma cells, to the 80% ethyl alcohol extract resulted in apoptotic cell death, fragmentation, downregulation in the expression levels of Bcl-XL, and upregulation of p53. The results indicated the inhibition of cell proliferation in a dose-dependent manner. The results highlight the activation of the mitochondrial apoptotic pathway in response to the treatment with ethanolic extracts of betel leaves. The treatment induced DNA damage leading to G1 cell cycle arrest and apoptosis. Low cytotoxicity and anticarcinogenic effects of the ethyl alcohol extracts of betel leaf on HT29 suggest an alternative approach to human colon cancer therapy (combination therapy) and highlights its significance as a functional food or food supplement.

Determination of apoptotic effects of newly synthesized avobenzone Sm(III) complex in non-small cell lung cancer

In this study, we aimed to investigate the apoptotic effects of the lanthanide complex TBK-23, which was used in biological imaging due to its magnetic and spectroscopic properties. The study utilized HTB-54 and BEAS-2B cell lines. The cells were subjected to TBK-23 and cisplatin at active doses for a duration of 24 hours. Subsequently, flow cytometry was employed to assess the quantity of apoptotic cells, while quantitative RT-PCR was used to measure the expression levels of BAX, BCL-2, and BCL-xL. While cisplatin at 31 µM increased the proapoptotic BAX expression level in HTB-54 and BEAS-2B cells, TBK-23 increased BAX in cancer cells at the same concentration, but there was no observable effect in healthy cells. TBK-23 was found to have higher inhibitory effect on the expression of the antiapoptotic BCL-2 gene in cancer cells compared to that of cisplatin-treated cells. Consequently, it was determined that TBK-23, a novel metal-based compound, exhibited apoptotic effects similar to that of cisplatin and could be a potential chemotherapeutic agent.

Potential effects of parietin on apoptosis and cell cycle related genes in SH-SY5Y neuroblastoma cells

Background: Ingredients obtained from natural products have been used in cancer treatments for years. High diversity and non-toxicity compared to chemotherapeutic agents are the main reasons for their preference. Lichens having potential for treatment of cancer consist of fungus and 1-2 species of algae. Under the name of lichen substances, many of them have also been synthesized as specific substances. The secondary metabolites in lichens are generally insoluble in water, and have many biological activities such as antiviral, antitumor, antibacterial, and antioxidant; they store in the fungal cell or on the surface of the hyphae and can only be extracted with organic solvents.&#x0D; Parietin extracted from lichen species such as xanthoria parietina is an anthraquinone pigment and a secondary metabolite. &#x0D; In our study, the effects of parietin on cytotoxicity, gene expression, migration, invasion, and colony formation in neuroblastoma cells treated with parietin were investigated. The SH-SY5Y neuroblastoma (NB), which is not treated with parietin, was administered as a control group.&#x0D; Methods: The IC50 value of the parietin was determined using XTT assay. The total RNA extractions were performed from the cells using the Tri-Reagent kit. The expressions of BCL-XL, BCL-2, MMP2, MMP9, P21, CYCLIN D1, CASAPASE-3, CASAPASE-9, BAX, P53, PUMA, and NOXA genes were investigated by Lightcycler 480 (Roche) using SYBR Green dye. Migration analysis of the control and the dose group cells were performed in accordance with the Wound-healing assay protocol. Invasion activities were determined using the “Invasion Chamber” (BD Biosciences) protocol. The colony assay was treated with crystal violet and the cells were observed under the light microscope.&#x0D; Results: The IC50 value of the parietin used for 48-hour treatment on the cells was determined as 35 µM. It was found that the expression levels of BCL-XL, BCL-2, MMP2, MMP9, P21, and CYCLIN D1 mRNA were downregulated, and it was also shown the expression levels of CASAPASE-3, CASAPASE-9, BAX, P53, PUMA, and NOXA to be upregulated. It was determined that parietin suppressed both cell invasion and migration, and colony formation in the neuroblastoma cells. &#x0D; Conclusions: Thus, it can be possible parietin to be used as an alternative, complementary, and supportive agent together with the other drugs in the treatment of neuroblastoma. However, more comprehensive studies supporting these significant effects of parietin will increase its potential in the application.

BCL-Xl Represents a Novel Therapeutic Target in Type 2 Mutant Calr-Driven Myeloproliferative Neoplasms

Myeloproliferative neoplasms (MPNs) are clonal disorders that arise in the hematopoietic stem cell (HSC) compartment and that result in the excess production of mature blood cells of the myeloid lineage. All MPNs are unified by their reliance on the constitutive activation of the JAK/STAT pathway. Nevertheless, JAK inhibitors have proven insufficient to significantly improve disease outcome, highlighting the need to further understand the molecular mechanisms driving these diseases and to discover novel therapeutic targets. Calreticulin (CALR), an endoplasmic reticulum (ER) chaperone protein, has been found to be mutated in ~40% of essential thrombocytosis (ET) and myelofibrosis (MF) cases. The most common mutations in CALR are type 1 (52 base pair deletion; CALRdel52) and type 2 (5 base pair insertion; CALRins5) mutations, which both confer distinct clinical and prognostic characteristics. We have previously demonstrated that mutant CALR-driven MPNs rely not only on aberrant JAK/STAT signaling but also on the upregulation of BCL-2 family proteins and activation of the unfolded protein response (UPR) pathway (ASH abstract #147859, 2021). Specifically, we have shown that CALRins5 mutations lead to activation of and dependency on the activating transcription factor 6 (ATF6) branch of the UPR. In parallel, we found that CALRins5 cells exhibit differential up-regulation of the anti-apoptotic protein BCL-xL, but not BCL-2 (ASH abstract #3588, 2021). Based on these findings, we hypothesized that the active form of ATF6, which functions as a transcription factor, may directly transcriptionally activate BCL-xL leading to its up-regulation specifically in CALRins5 but not CALRdel52 MPN cells, and that the ATF6/BCL-xL axis may represent an important pro-survival mechanism that can be targeted for therapeutic gain in CALRins5 MPN patients. To test this hypothesis, we first validated that BCL-xL is significantly up-regulated in CALRins5 versus CALRdel52 and CALR wild type (CALRwt) human cell lines and peripheral blood mononuclear cells (PBMCs) from MPN patients. Because BCL-xL can also be regulated downstream of JAK/STAT signaling, we next tested whether the increased expression of BCL-xL in CALRins5 cells is due to ATF6 activation, JAK/STAT activation, or both. In cells treated with JAK inhibitor ruxolitinib, we found that while BCL-xL expression levels decreased nearly 90% in ruxolitinib treated CALRdel52 cells, this decrease was much more modest in CALRins5 cells, suggesting JAK/STAT activation is only partially responsible for BCL-xL up-regulation in CALRins5 MPN cells. Conversely, in ATF6 knockout cells, we found that deletion of ATF6 did not affect BCL-xL levels in CALRwt or CALRdel52 cells, but did significantly abrogate BCL-xL up-regulation in CALRins5 cells. This suggests that in CALRins5 cells, while JAK/STAT signaling contributes in part to BCL-xL up-regulation, ATF6 is the key mediator of BCL-xL expression. CUT&amp;RUN studies further revealed that ATF6 directly regulates BCL-xL transcription. Lastly, we sought to test whether inhibition of BCL-xL alone is sufficient to induce apoptosis in CALRins5 cells. BH3 profiling studies revealed that CALRins5 cells are more primed for apoptosis via BCL-xL peptide antagonism compared to CALRdel52 cells. We next evaluated the activity of A-1331852, a BCL-xL selective inhibitor, alone or in combination with ruxolitinib, in human cell lines and PMBCs from MPN patients. We observed that CALRins5 cells displayed decreased viability in response to A-1331852 alone and in combination treatments, suggesting an increased sensitivity to BCL-xL inhibition. In conclusion, we demonstrate that CALRins5-driven MPN cells display an enhanced sensitivity to BCL-xL inhibition, which may represent an effective therapeutic approach for CALRins5+ MPN patients.

Human papillomavirus maybe is a critical player in the regulation of chemoresistance related factors (P53, Rb, TWIST, Bcl-2, Bcl-XL, c-IAP2, cytochrome C, and caspase 3) in breast cancer

As one of the frequent malignancies, breast cancer (BCa) is the foremost reason for cancer-related deaths among women. The role of Human papillomavirus (HPV) in chemoresistance has rarely been investigated in previous studies. The current study sets out to the possible role of HPV in BCa chemoresistance. In this research, 90 BCa tissue and 33 normal breast tissue were collected. We evaluated the presence of the HPV genome along with the viral (E2, E6, E7) and cellular gene expression associated with cell resistance to death. Statically significant differences in the prevalence of HPV between the BCa group (25.2% or 23/90) and the control group (21.8% or 7/32) were not found. HPV-16 and HPV-18 genotypes were the abundant HPV genotypes. Resistance to the Adriamycin-Cyclophosphamide (AC), paclitaxel regimen was elevated in the HPV- group (56/70) in comparison to the HPV+ group (14/70). Nevertheless, there was no significant difference in the prevalence of resistance to AC + paclitaxel + triple-negative breast cancer combination therapy between the HPV+ group (9/20) and in the HPV- group (11/20). In the BCa group in contrast to the control group, the expression level of Bcl-2, BCL-XL, and c-IAP2 demonstrated a significant decrease, while, the expression level of cytochrome C and caspase 3 was significantly increased. This study suggests that HPV infection might contribute to BCa chemoresistance through disrupt cellular genes involved in cell death.

Abstract 4673: Therapeutic potential of polatuzumab vedotin for the treatment of diffuse large B-cell lymphoma

Abstract Background Diffuse large B-cell lymphoma (DLBCL) is typically treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). Although most patients can be cured with R-CHOP, up to one-third of them relapses with a dismal outcome in most cases. Numerous approaches have been attempted to improve the treatment outcomes with R-CHOP. Polatuzumab vedotin is an antibody-drug conjugate targeting CD79b, which is ubiquitously expressed on over 90% of B-cell NHL malignancies, including DLBCL. To determine the potential of polatuzumab vedotin as a therapeutic agent, we evaluate its potency across a series of DLBCL patient derived xenograft (PDX) models. Method We established and characterized a series of DLBCL lymphoma PDX models. Eight DLBCL PDX models were selected for polatuzumab vedotin efficacy study, of which three are GCB DLBCL. Gene expression analysis of these lymphoma PDX models was performed using an Illumina NovaSeq 6000 system following Illumina-provided protocols for 2 × 150 paired-end sequencing. The expression status of CD79b on these DLBCL PDX models was also evaluated by IHC. Conclusion Our data showed that a single dose of polatuzumab vedotin at just 2 mg/kg could reached a comparable effect with R-CHOP and no significant body weight loss was observed. Our result showed that there is no apparent correlation between polatuzumab vedotin responses (TGI) and the CD79b expression (IHC Score). Although polatuzumab vedotin demonstrated encouraging activity in the treatment of DLBCL, some of the PDX models are also resistant to polatuzumab vedotin treatment. To further investigate the molecular parameters of the relative sensitivity of polatuzumab vedotin. Gene expression profiling of those PDX models and their sensitivity to polatuzumab vedotin were conducted, we found that the expression level of BCL-XL was correlated with reduced sensitivity to polatuzumab vedotin. Citation Format: Hui Qi, Fuyang Wang, Bingrui Han, Xiaomin Wang, Xiangnan Qiang, Zhixiang Zhang, Qiangyang Gu. Therapeutic potential of polatuzumab vedotin for the treatment of diffuse large B-cell lymphoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4673.

The anti-apoptosis effect of isovitexin on human keratinocytes by regulating miR-98-5p/Bcl-2/Bcl-xL and MAPKs/NF-κB signaling pathways

Isovitexin is a highly bioactive food-derived flavonoid currently attracting attention for its signal transduction-based antioxidant effects. This study focused on the potential molecular mechanisms by which isovitexin protects HaCaT cells from oxidation-induced apoptosis. It was found that isovitexin significantly reversed oxidative stress-induced apoptosis by reducing ROS level. In addition, isovitexin inhibited apoptosis in a dose-dependent manner by upregulating the expression level of Bcl-xL and Bcl-2 and downregulating the expression level of Bax and caspase-3. It is important to note that the effect of isovitexin may be achieved by targeting miR-98-5p, an upstream target of Bcl-xL and Bcl-2, and overexpression of miR-98-5p counteracted isovitexin-mediated upregulation of antiapoptotic proteins. Isovitexin also inhibited apoptosis by reducing the level of IL-6, IL-1β and TNF-α, which was achieved by regulating MAPKs/NF-κB signaling pathway. Overall, this study confirmed the mechanism of multi-targeted inhibition of apoptosis by targeting miR-98-5p/Bcl-2/Bcl-xL and MAPKs/NF-κB signaling pathway by isovitexin.

Abstract P3-11-01: Increasing DAXX Expression in ER+ Breast Cancer Cells to Overcome Endocrine Therapy Resistance

Abstract Background: New treatment paradigms are needed to overcome resistance to endocrine therapy (ET; tamoxifen or aromatase inhibitors, AI) in ER+ breast cancer (BC). ET resistance is due to survival of breast cancer stem cells (BCSCs) that contribute to relapse of ER+ BC. Notch signaling drives BCSCs. In order to identify Notch specific biomarkers for the purpose of patient selection for anti-Notch therapy, we conducted a pre-surgical biomarker window study combining ET plus MK-0752, a -secretase inhibitor (GSI). Death Associated Protein 6 (DAXX) was discovered to be a novel Notch1 target gene and necessary for GSI-mediated inhibition of BCSCs. Subsequently, we found that DAXX alone was sufficient to inhibit BCSCs. In this current study, we investigated the mechanism by which high DAXX expression inhibited growth of ET resistant ER+ BC cells in vitro and in vivo. Methods: Isogenic ER+ BC cell lines (parental MCF-7, ET resistant MCF-7/5C, parental T47D, and ET resistant T47D-ED) were used. Cells were cultured in estrogen-deprived medium for more than 1 year to mimic AI use. DAXX was depleted using siRNA or overexpressed using a pCMV-expression vector. Bulk cell proliferation was analyzed in response to estrogen depletion or increasing concentrations of 17-estradiol. BCSC survival was measured using the mammosphere-forming assay. Tumor onset and burden were measured by injecting DAXX-expressing or depleted mammospheres into mammary fat pads of female, athymic nude mice. Recurrence of an ER+ PDX tumor (BCM 5097) was measured after withdrawal of estrogen. RNA sequencing identified enriched genes and pathways that required DAXX. Based on these results, cell death was assessed using Annexin V/7-AAD flow cytometry, PARP-1 and Caspase 8 cleavage, phosphorylation of JNK, and expression of apoptotic protein regulators, BIM, BAX, Bcl-2, and Bcl-xL. JNK signaling was inhibited using SB600125. Results: Estradiol stimulated proliferation of parental, ER+ MCF-7 and T47D cells. In contrast, estradiol inhibited proliferation of isogenic ET resistant MCF-7/5C and T47D-ED cells in a concentration and ER-dependent manner. Estradiol induced the DAXX protein in both ET sensitive and resistant cells. DAXX was required for BCSC survival in ET sensitive cells. However, once ER+ cells acquired resistance to ET, DAXX was necessary and sufficient to inhibit both bulk cell proliferation and BCSC survival, suggesting that increasing DAXX might be a novel approach to overcome ET resistance. In mice, high DAXX expression significantly inhibited tumor onset and burden of ET resistant tumors compared to DAXX-depleted tumors. Low DAXX expression was significantly associated with recurrence of an ER+ PDX tumor (BCM 5097) after withdrawal of estrogen. RNA sequencing revealed that DAXX activated an anti-neoplastic gene signature, including transcription factors that regulate cell death genes including the Bcl2-family. DAXX was required for high BIM expression and low levels of Bcl-xL. DAXX was necessary and sufficient to induce apoptosis, PARP-1 cleavage, and phosphorylation of JNK in ET resistant cells. A selective JNK inhibitor, SB600125 rescued DAXX-mediated inhibition of ET resistant bulk cell proliferation and BCSC survival, suggesting that high DAXX expression activates JNK signaling to regulate apoptotic proteins to induce cell death of BCSCs-derived from ET resistant BC. Conclusions: Expressing high DAXX levels is a potent method to inhibit ET-resistant BC cell proliferation and BCSC survival. The mechanism by which DAXX inhibits ET-resistant BC is through activation of JNK signaling, regulation of pro-apoptotic genes, and induction of apoptosis. The translational impact of this research is to identify novel agents that can increase DAXX expression and test them pre-clinically and in clinical trials for patients with ET-resistant breast cancer. Citation Format: Clodia Osipo, Kathy S. Albain, Daniel Peiffer, Debra Wyatt. Increasing DAXX Expression in ER+ Breast Cancer Cells to Overcome Endocrine Therapy Resistance [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-11-01.

Effects of epigenetic modifier on the developmental competence and quantitative expression of genes in male and female buffalo (Bubalus bubalis) cloned embryos.

Adult male and female Murrah buffalo fibroblast cells were used as donors for the production of embryos using handmade cloning. Both donor cells and reconstructed embryos were treated with 50 nM trichostatin-A (TSA) and 7.5 nM 5-aza-2'-deoxycytidine (5-aza-dC). The blastocyst rate of both treated male (40.1% ± 2.05) and female (37.0% ± 0.83) embryos was significantly lower than in untreated control males (49.7% ± 3.80) and females (47.2% ± 2.44) but their apoptotic index was lower (male, control: 5.90 ± 0.48; treated: 4.96 ± 0.31): (female, control: 8.11 ± 0.67; treated: 6.65 ± 0.43) and epigenetic status in terms of global acetylation and methylation of histone was significantly improved. The expression level of hypoxanthine-guanine phosphoribosyltransferase (HPRT) was higher (P < 0.05) and that of PGK, G6PD, OCT 4, IFN-tau and CASPASE3 was significantly lower (P < 0.05) in treated male blastocyst than control and the expression levels of DNMT1, IGF1R and BCL-XL were not significantly different between the two groups. In the female embryos, the relative mRNA abundance of OCT4 was significantly higher (P < 0.05), and that of XIST and CASPASE3 was significantly lower (P < 0.05) in the epigenetic modifier-treated group compared with that of the control group, whereas the expression levels of HPRT, PGK, G6PD, DNMT1, IFN-tau, IGF1R and BCL-XL were not significantly different between the two groups. In both embryos, a similar effect of treatment was observed on genes related to growth and development, but the effect on the expression of X-linked genes varied. These results indicate that not all X-linked genes respond to TSA and 5-aza-dC treatment in the same manner.

Hyperforin Elicits Cytostatic/Cytotoxic Activity in Human Melanoma Cell Lines, Inhibiting Pro-Survival NF-κB, STAT3, AP1 Transcription Factors and the Expression of Functional Proteins Involved in Mitochondrial and Cytosolic Metabolism.

Hyperforin (HPF), the main component responsible for the antidepressant action of Hypericum perforatum, displays additional beneficial properties including anti-inflammatory, antimicrobic, and antitumor activities. Among its antitumor effects, HPF activity on melanoma is poorly documented. Melanoma, especially BRAF-mutated melanoma, is still a high-mortality tumor type and the currently available therapies do not provide solutions. We investigated HPF's antimelanoma effectiveness in A375, FO1 and SK-Mel-28 human BRAF-mutated cell lines. Cell viability assays documented that all melanoma cells were affected by low HPF concentrations (EC50% 2-4 µM) in a time-dependent manner. A Br-deoxy-uridine incorporation assay attested a significant reduction of cell proliferation accompanied by decreased expression of cyclin D1 and A2, CDK4 and of the Rb protein phosphorylation, as assessed by immunoblots. In addition, the expression of P21/waf1 and the activated form of P53 were increased in A375 and SK-Mel-28 cells. Furthermore, HPF exerts cytotoxic effects. Apoptosis is induced 24 h after HPF administration, documented by an increase of cleaved-PARP1 and a decrease of both Bcl2 and Bcl-xL expression levels. Autophagy is induced, attested by an augmented LC3B expression and augmentation of the activated form of AMPK. Moreover, HPF lowers GPX4 enzyme expression, suggesting ferroptosis induction. HPF has been reported to activate the TRPC6 Ca++ channel and/or Ca++ and Zn++ release from mitochondria stores, increasing cytosolic Ca++ and Zn++ concentrations. Our data highlighted that HPF affects many cell-signaling pathways, including signaling induced by Ca++, such as FRA1, pcJun and pCREB, the expression or activity of which are increased shortly after treatment. However, the blockage of the TRPC6 Ca++ channel or the use of Ca++ and Zn++ chelators do not hinder HPF cytostatic/cytotoxic activity, suggesting that damages induced in melanoma cells may pass through other pathways. Remarkably, 24 h after HPF treatment, the expression of activated forms of the transcription factors NF-κB P65 subunit and STAT3 are significantly lowered. Several cytosolic (PGM2, LDHA and pPKM2) and mitochondrial (UQCRC1, COX4 and ATP5B) enzymes are downregulated by HPF treatment, suggesting a generalized reduction of vital functions in melanoma cells. In line with these results is the recognized ability of HPF to affect mitochondrial membrane potential by acting as a protonophore. Finally, HPF can hinder both melanoma cell migration and colony formation in soft agar. In conclusion, we provide evidence of the pleiotropic antitumor effects induced by HPF in melanoma cells.