Venetoclax-Resistant MV4-11 Leukemic Cells Activate PI3K/AKT Pathway for Metabolic Reprogramming and Redox Adaptation for Survival.

Venetoclax (ABT199) is a selective B-cell lymphoma 2 (BCL-2) inhibitor. The US FDA recently approved it to be used in combination with low-dose cytarabine or hypomethylating agents in acute myeloid leukemia (AML) or elderly patients non-eligible for chemotherapy. However, acquiring resistance to venetoclax in AML patients is the primary cause of treatment failure. To understand the molecular mechanisms inherent in the resistance to BCL-2 inhibitors, we generated a venetoclax-resistant cell line model and assessed the consequences of this resistance on its metabolic pathways. Untargeted metabolomics data displayed a notable impact of resistance on the PI3K/AKT pathway, the Warburg effect, glycolysis, the TCA cycle, and redox metabolism. The resistant cells showed increased NADPH and reduced glutathione levels, switching their energy metabolism towards glycolysis. PI3K/AKT pathway inhibition shifted resistant cells towards oxidative phosphorylation (OXPHOS). Our results provide a metabolic map of resistant cells that can be used to design novel metabolic targets to challenge venetoclax resistance in AML.

Highly Expressed DLL4 and JAG1: Their Role in Incidence of Breast Cancer Metastasis.

BACKGROUND: The role of Notch signaling dysregulation in causing metastatic breast cancer is not yet elucidated, therefore, this study aimed to investigate the expression of DLL4 and JAG1 in metastatic breast cancer. Moreover, we examined the possible association between clinicopathological features and studied parameters. DESIGN AND METHODS: A total of 90 patients with invasive ductal breast carcinomas (52 non-metastatic and 38 metastatic) were enrolled in the current study. Furthermore, there were 42 patients with benign breast diseases. The mRNA and protein expression of DLL4 and JAG1 were analyzed by RT-PCR and ELISA, respectively in breast cell lysates. RESULTS: The mRNA and protein expression of DLL4 and JAG1 were obviously higher in patients with breast cancer compared to patients with benign breast diseases and in metastatic versus non-metastatic breast cancer. A significant positive correlation was declared between DLL4 and JAG1 at both mRNA and protein levels in metastatic and localized breast cancer patients. Highly expressed mRNA and protein of DLL4 and JAG1 were associated with late tumor stages; moreover, upregulation of mRNA and protein of JAG1 was correlated with poorly differentiated tumors. CONCLUSION: Our data emphasize that overexpression of DLL4 and JAG1 could predict the development of distant metastasis in breast cancer patients.

Concomitance of downregulated active caspase-3 and upregulated X-chromosome linked inhibitor of apoptosis protein as a sensitive diagnostic approach for breast cancer.

We aimed to explore the efficacy of active caspase-3 and X-chromosome linked inhibitor of apoptosis protein (XIAP) as diagnostic markers for breast cancer. Furthermore, we examined the relationship between the examined parameters and clinicopathological factors. The current study involved 96 patients diagnosed with breast cancer and 40 patients had benign breast diseases. The expression of active caspase-3 was analyzed by both ELISA and Western blot, whereas the expression of XIAP was determined by ELISA in cell lysates. Active caspase-3 was significantly downregulated, while XIAP was markedly upregulated in patients with breast cancer in comparison to benign group. A significant negative correlation was observed between active caspase-3 and XIAP in breast cancer patients. Low active caspase-3 expression was associated with high grade, whereas, the high XIAP level was correlated with poorly differentiated tumors and late tumor stages. The sensitivity and specificity were 73.96% and 80.0% for active caspase-3, and, 70.83% and 82.5% for XIAP. A combination of active caspase-3 and XIAP provided a promising sensitivity of 88.54% and specificity of 90.0%. Our data indicate that active caspase-3 and XIAP could be substantial diagnostic markers for breast cancer patients.

p21Waf1/Cip1: its paradoxical effect in the regulation of breast cancer.

p21Waf1/Cip1, the cyclin-dependent kinase (CDK) inhibitor belonging to the KIP/CIP family, was initially regarded as a tumor suppressor protein because it was recognized as the chief mediator of p53-dependent cell cycle arrest elicited by DNA damage. Conversely, it has been proposed that p21Waf1/Cip1 may also function as an oncogene because it can inhibit apoptosis. Thus, p21Waf1/Cip1 is regarded as a protein with a dual behavior, as its expression might cause potential benefits or dangerous effects in breast cancer. Consequently, careful planning is required in targeting p21Waf1/Cip1 expression for therapy of breast cancer patients. This review illustrates the discovery and mechanisms of induction of p21Waf1/Cip1. Then, we focus on elucidating the paradoxical effect of p21Waf1/Cip1 expression on human breast carcinogenesis and explaining how the subcellular localization (nuclear or cytoplasmic) of p21Waf1/Cip1 has an impact on both determining its fate as either cell-growth inhibitor or antiapoptotic molecule and, its effect on clinicopathological factors and prognosis of breast cancer patients. Moreover, we explore how the pattern of the p21Waf1/Cip1 could affect the responsiveness of human breast cancer to chemotherapy. Furthermore, the pharmacological approaches to target p21Waf1/Cip1 expression for therapy of breast cancer are clarified.

The KIP/CIP family members p21

OBJECTIVE: We examined the expression status of p21^{Waf1/Cip1} and p57^{Kip2} in breast cancer as well as their relationship with clinicopathological factors. Moreover, the diagnostic value of gene promoter methylation of p21^Waf1/Cip1 and p57^Kip2 was assessed in breast cancer patients. METHODS: This study involved 85 patients diagnosed with breast cancer and 36 patients with benign breast lesions. The expression of p21^{Waf1/Cip1} and p57^{Kip2} in cell lysates was analyzed by ELISA and Western blot, respectively. The gene promoter methylation of p21^Waf1/Cip1 and p57^Kip2 was examined in cell lysates by methylation specific PCR. RESULTS: p21^{Waf1/Cip1} expression was higher while p57^{Kip2} level was lower in breast cancer patients compared to patients with benign breast lesions. The combined use of p21^{Waf1/Cip1} and p57^{Kip2} provided sensitivity and specificity of 82.35% and 86.11%, respectively. None of the malignant and benign breast tumors were found to be hypermethylated at p21^Waf1/Cip1 gene promoter. However, aberrant methylation of p57^Kip2 gene promoter was detected in 49 of 85 (57.65%) of breast cancer tumors. High p21^{Waf1/Cip1} level was associated with high grade, late stages and lymph node involvement, whereas low p57^{Kip2} level was correlated with high grade and HER2 overexpressing breast cancer. Moreover, hypermethylated p57^Kip2 gene promoter was associated with high grade. CONCLUSION: Our findings show that the overexpression of p21^{Waf1/Cip1}, down-expression of p57^{Kip2} and gene promoter methylation of p57^Kip2 could be considered as promising diagnostic markers for breast cancer.

Circulating Fibroblast Growth Factor-23 Level and Paraoxonase-1 Lactonase Activity in Chronic Hemodialysis Patients: Their Impact on the Incidence of Native AV Fistula Thrombosis.

PURPOSE: Thrombosis of native arteriovenous (AV) fistula is an important cause of complications in hemodialysis (HD) patients. The purpose of this study was to investigate the usefulness of measuring circulating fibroblast growth factor-23 (FGF-23) level and paraoxonase-1 (PON1) lactonase activity as potential predictors of native AV fistula thrombosis in chronic HD patients. METHODS: This study included 83 HD patients (48 with thrombosed and 35 with non-thrombosed native AV fistulas) and 38 healthy volunteers. Serum FGF-23 level was measured using the ELISA technique, while serum PON1 lactonase activity was measured spectrophotometrically using gamma-thiobutyrolactone as a substrate. RESULTS: FGF-23 was significantly increased while PON1 lactonase was markedly decreased in both thrombosed and non-thrombosed HD patients compared with controls (P < 0.001). FGF-23 was elevated whereas PON1 lactonase was decreased in HD patients with thrombosed native AV fistulas compared with HD patients with non-thrombosed native AV fistulas (P = 0.001 and 0.002, respectively). A significant negative correlation was found between FGF-23 and PON1 lactonase in HD patients with thrombosed native AV fistulas (r = -0.342, P = 0.017). CONCLUSIONS: This study shows a potential value of FGF-23 and PON1 lactonase as predictors of native AV fistula thrombosis in HD patients.

Urinary retinoic acid receptor-ß2 gene promoter methylation and hyaluronidase activity as noninvasive tests for diagnosis of bladder cancer.

OBJECTIVES: We evaluated the significance of urinary retinoic acid receptor-ß2 (RAR-ß2) gene promoter methylation and hyaluronidase activity in comparison with voided urine cytology (VUC) in diagnosis of bladder cancer. DESIGN AND METHODS: This study included 100 patients diagnosed with bladder cancer, 65 patients with benign urological disorders and 51 healthy volunteers. Urine supernatant was used for determining hyaluronidase activity by zymography while urine sediment was used for cytology and detection of methylated RAR-ß2 gene promoter by methylation specific nested PCR. RESULTS: The sensitivity and specificity were 53% and 90.5% for VUC, 65% and 89.7% for percent methylation fraction of RAR-ß2 gene promoter, and 89% and 90.5% for hyaluronidase activity; combination of the three parameters increased sensitivity to 95%. A significant association was observed between investigated markers and advanced grade tumor. CONCLUSIONS: Combined use of RAR-ß2 gene promoter methylation, hyaluronidase activity and VUC is promising non-invasive tool for bladder cancer detection.

Significance of survivin and Bcl-2 homologous antagonist/killer mRNA in detection of bone metastasis in patients with breast cancer.

The aim of this study was to assess the diagnostic value of survivin and Bcl-2 homologous antagonist/killer (Bak) mRNA in the detection of patients with bone metastatic breast cancer. This study included 92 patients with breast carcinoma (54 non-metastatic and 38 bone metastatic) and 31 patients with benign breast lesions. Survivin in cell lysates was measured by ELISA while tissue Bak mRNA was detected by RT-PCR. Survivin was significantly increased in bone metastatic breast cancer patients compared to non-metastatic cases or benign ones. Bak mRNA was markedly decreased in bone metastatic patients compared to non-metastatic ones, while significant expression of Bak mRNA was observed in bone metastatic cases compared to benign patients. High survivin level was associated with high grade, late stages and lymph node involvement, whereas low Bak mRNA was associated with late stages. Our data indicate that survivin and Bak mRNA were considerable markers for the identification of breast cancer patients with bone metastasis.

Clinical utility of circulating matrix metalloproteinase-7 (MMP-7), CC chemokine ligand 18 (CCL18) and CC chemokine ligand 11 (CCL11) as markers for diagnosis of epithelial ovarian cancer.

Ovarian cancer remains a highly lethal disease. The aim of the present study was to evaluate the usefulness of measuring serum matrix metalloproteinase-7 (MMP-7), CC chemokine ligand 18 (CCL18) and CC chemokine ligand 11 (CCL11) in comparison with serum cancer antigen 125 (CA 125) for diagnosis of epithelial ovarian cancer (EOC). This study included 51 patients with EOC, 27 patients with benign ovarian lesions and 29 healthy volunteers. Serum CA 125 was determined by microparticle enzyme immunoassay, while serum MMP-7, CCL18 and CCL11 were measured using enzyme-linked immunosorbent assay. The sensitivity and specificity were 86.3% and 92.9% for CA 125, 80.4% and 87.5% for MMP-7, 84.3% and 91.1% for CCL18 and, 68.6% and 62.5% for CCL11. Combination of CA 125, MMP-7, CCL18 and CCL11 gave a promising sensitivity of 100%, but specificity was decreased to 60.7%. The combined use of serum CA 125, MMP-7, CCL18 and CCL11 effectively detected early stages EOC with high sensitivity of 94.4%. Our data indicate that serum MMP-7, CCL18 and CCL11, in combination with CA 125 could be useful in diagnosis of EOC.

Diagnostic value of fibronectin and mutant p53 in the urine of patients with bladder cancer: impact on clinicopathological features and disease recurrence.

Development of new methods for bladder cancer detection is required because cystoscopy is invasive, and voided urine cytology (VUC) has low sensitivity. The aim of this study was to evaluate the diagnostic performance of urinary fibronectin and mutant p53 in comparison with VUC in the detection of bladder cancer. This study included 100 patients diagnosed with bladder cancer, 93 patients with benign urological disorders and 47 healthy volunteers. The urine supernatant was used for determination of fibronectin by ELISA, while urine sediment was used for cytology and detection of mutant p53 by PCR-SSCP followed by DNA sequencing. The sensitivity and specificity were 59% and 91.4% for VUC, 82% and 84.3% for fibronectin, and 37% and 100% for mutant p53; combination of the three parameters increased sensitivity to 95% but specificity was only 78.6%. A significant association was observed between disease recurrence and mutant p53, stage and lymph node involvement. Our results indicate that fibronectin had the highest sensitivity compared to VUC and mutant p53 in bladder cancer detection; however, mutant p53 had superior specificity compared to VUC and fibronectin. Mutant p53 is associated with disease recurrence and hence it has a significant prognostic role in bladder cancer.

Comparison of CD44 and cytokeratin 20 mRNA in voided urine samples as diagnostic tools for bladder cancer.

OBJECTIVES: We evaluated the diagnostic efficacy of urinary CD44 and cytokeratin 20 (CK20) mRNA in comparison with voided urine cytology (VUC) for the detection of bladder cancer. DESIGN AND METHODS: A total of 136 Egyptian patients provided a single voided urine sample for CD44, CK20 mRNA and VUC before cystoscopy. Of the 136 cases, 111 were histologically diagnosed as bladder cancer whereas the remaining 25 had benign urological disorders. A group of 20 healthy volunteers was also included in this study. Voided urine was centrifuged and the urine sediment was used for cytology, estimation of CD44 by ELISA and RNA extraction. CK20 mRNA was detected by conventional RT-PCR and quantitative real-time RT-PCR. RESULTS: The best cutoff values for CD44 and relative CK20 mRNA detected by real-time RT-PCR were calculated by receiver operating characteristic curve. The positivity rates and the mean ranks for CD44 and CK20 mRNA showed significant difference among the three investigated groups (p=0.001). Quantitative real-time RT-PCR results were comparable to conventional RT-PCR for the detection of CK20 mRNA. The positivity rate of CD44 was significantly associated with schistosomiasis and urine cytology. The overall sensitivity and specificity were 52.3% and 88.9% for VUC, 63.1% and 88.9% for CD44, and 82.0% and 97.8% for CK20 mRNA. Combined sensitivity of VUC with CD44 and CK20 mRNA together (95.5%) was higher than either the combined sensitivity of VUC with CD44 (78.4%) or with CK20 mRNA (91.0%) or than that of the biomarker alone. CONCLUSION: Urinary CD44 and CK20 mRNA had higher sensitivities compared to VUC. However, when the diagnostic efficacy was considered, CK20 mRNA by either conventional RT-PCR or real-time RT-PCR had the highest sensitivity and specificity compared to CD44 and VUC.

Evaluation of circulating vascular endothelial growth factor and soluble adhesion molecules as reliable predictors of native arteriovenous fistula thrombosis in chronic hemodialysis patients.

OBJECTIVES: We evaluated the possibility of using circulating vascular endothelial growth factor (VEGF) and soluble adhesion molecules as reliable predictors of native arteriovenous (AV) fistula thrombosis in chronic hemodialysis (HD) patients. DESIGN AND METHODS: This study included 62 HD patients (34 with thrombosed and 28 with non-thrombosed AV fistulas) and 21 healthy volunteers. Serum VEGF, soluble vascular cell adhesion molecule-1 (sVCAM-1), soluble intercellular adhesion molecule-1 (sICAM-1), and soluble E-selectin (sE-selectin) were measured using ELISA technique. RESULTS: VEGF, sVCAM-1, sICAM-1 and sE-selectin median levels were higher in HD patients compared to controls (p=0.000 for all parameters). Increased median levels of VEGF and sVCAM-1 were demonstrated in HD patients with thrombosed AV fistulas compared to HD patients with non-thrombosed AV fistulas (p=0.003 and 0.000, respectively). A significant positive correlation has been found between VEGF and sVCAM-1 in HD patients with thombosed AV fistulas (r=0.525, p=0.001). CONCLUSIONS: The assessment of serum VEGF and sVCAM-1 might be useful for the identification of the chronic HD patients at an increased risk for native AV fistulas thrombosis. The clinical relevance of these observations warrants further investigations.

The diagnostic efficacy of urinary TGF-beta1 and VEGF in bladder cancer: comparison with voided urine cytology.

The purpose of this study was to evaluate the diagnostic efficacy of urinary transforming growth factor-beta1 (TGF-beta1) and vascular endothelial growth factor (VEGF) in comparison with voided urine cytology in the detection of bladder cancer. This study included 120 patients with bladder cancer, 54 patients with benign urological disorders and 55 healthy volunteers. Urine supernatant was used for estimation of TGF-beta1 and VEGF by ELISA. VEGF was detected by Western blot (WB) analysis in the urine supernatant of randomly selected bladder cancer patients. The urine sediment was used for cytology. There was a statistically significant difference in the median levels of TGF-beta1 (P=0.002) and VEGF (P=0.000) between the control, benign and malignant groups. The concordance rate of VEGF ELISA with VEGF WB was 96.3%. The overall sensitivity and specificity were 70.8% and 90.8% for voided urine cytology, 71.6% and 59.6% for TGF-beta1, and 76.7% and 61.5% for VEGF. The combined use of voided urine cytology with TGF-beta1 and VEGF improved the sensitivity up to 94.9%, although it lowered specificity to 62.0%. There was a significant association between positivity rate of TGF-beta1 and positive urine cytology samples (P=0.023). Median level and positivity rate of VEGF were significantly associated with early stage (I, II) of bladder carcinoma (P=0.01 and 0.025, respectively). Our data indicate that urinary TGF-beta1 and VEGF had higher sensitivities compared to voided urine cytology. Moreover, the combined sensitivity of voided urine cytology with TGF-beta1 and VEGF together was higher than sensitivity of voided urine cytology alone in detection of bladder cancer.

Predictive value of osteocalcin and beta-CrossLaps in metastatic breast cancer.

OBJECTIVES: Assessment of the diagnostic value of serum CEA, CA 15.3, osteocalcin (OC) and beta-CrossLaps (beta-CTX) in the detection of metastatic breast cancer. DESIGN AND METHODS: This study included 47 patients with breast cancer (20 non-metastatic breast cancer, 11 bone metastasis, 11 soft tissue metastasis, 5 bone plus soft tissue metastasis), 10 patients with benign breast lesions and 13 healthy volunteers. CEA and CA 15.3 were determined using microparticle enzyme immunoassay; while OC and beta-CTX were measured by electrochemiluminescence immunoassay. RESULTS: CEA, CA 15.3, OC and beta-CTX median levels were higher in breast cancer patients compared to controls (p=0.006, 0.001, 0.004 and 0.038, respectively). Increased levels of OC and beta-CTX were demonstrated in bone metastatic patients compared to non-metastatic or soft tissue metastatic patients (p=0.000). CONCLUSIONS: Combined use of OC and beta-CTX could be useful in early detection of bone metastatic breast cancer which might improve the outcome of the disease.

Antibody to E1 peptide of hepatitis C virus genotype 4 inhibits virus binding and entry to HepG2 cells in vitro.

AIM: To analyze the neutralizing activity of antibodies against E1 region of hepatitis C virus (HCV). Specific polyclonal antibody was raised via immunization of New Zealand rabbits with a synthetic peptide that had been derived from the E1 region of HCV and was shown to be highly conserved among HCV published genotypes. METHODS: Hyper-immune HCV E1 antibodies were incubated over night at 4 degree Celsius with serum samples positive for HCV RNA, with viral loads ranging from 615 to 3.2 million IU/ mL. Treated sera were incubated with HepG2 cells for 90 min. Blocking of viral binding and entry into cells by anti E1 antibody were tested by means of RT-PCR and flow cytometry. RESULTS: Direct immunostaining using FITC conjugated E1 antibody followed by Flow cytometric analysis showed reduced mean fluorescence intensity in samples pre-incubated with E1 antibody compared with untreated samples. Furthermore, 13 out of 18 positive sera (72%) showed complete inhibition of infectivity as detected by RT-PCR. CONCLUSION: In house produced E1 antibody, blocks binding and entry of HCV virion infection to target cells suggesting the involvement of this epitope in virus binding and entry. Isolation of these antibodies that block virus attachment to human cells are useful as therapeutic reagents.