[Clinicopathological characteristics of angioimmunoblastic T-cell lymphoma with B-cell proliferation or neoplasms].

Objective: To investigate the clinical, pathological and immunophenotypic features, and differential diagnosis of angioimmunoblastic T-cell lymphoma (AITL) with B-cell proliferation or neoplasms. Methods: Eight qualified cases were collected from the Department of Pathology, the First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China from January 2019 to July 2023. One case was diagnosed with AITL and diffuse large B-cell lymphoma (DLBCL) and the other seven cases were diagnosed with AITL and B-cell proliferation. Clinical characteristics and pathological morphology were summarized. Immunohistochemical analysis, fluorescence in situ hybridization and gene rearrangement detection were performed. Results: The patients' average age was 58 years. Five of them were male. Biopsies of the enlarged cervical lymph nodes showed structural destruction and exhibited various histologic patterns. Some cases revealed Burkitt-like morphology, a moderate tumor volume and slightly irregular nuclei. Some cases showed prominent nucleoli. High endothelial venules and expanded follicular dendritic cells were detected. Tumor cells derived from T-follicular helper (TFH) cells were positive for two or more TFH biomarkers. Nodular or diffuse patchy proliferation of B cells was noted around the tumor tissue, which was initially considered as B-cell lymphoma. All of the 8 cases showed monoclonal rearrangements of the T-cell receptor genes while 5 of them also showed clonal rearrangements of the Ig genes. Seven of the 8 cases were subject to the detection of C-MYC gene breakage and were all negative. EBV-positive cells were seen in 6 cases. Neoplastic B cells were positive for C-MYC (>40%), while proliferative B cells were negative for C-MYC (<40%). Conclusions: The histological morphology of AITL with B-cell proliferation or lymphoma may be different from AITL. An integrated analysis, incorporating clinical, morphologic, immunophenotypic, and molecular assessment, helps reach an accurate diagnosis. This group of cases demonstrated the clinical and pathological characteristics of AITL accompanied by B-cell proliferation and B-cell lymphoma. The findings suggest that C-MYC maybe a feasible indicator for distinguishing B-cell proliferation from B-cell lymphoma, and provide a simple and feasible immunohistochemical marker for the diagnosis and research of composite lymphoma.

[Clinicopathological features and prognosis of sporadic mismatch repair deficient colorectal cancer].

Objective: To investigate the clinicopathological characteristics and prognostic factors of sporadic mismatch repair deficient (dMMR) colorectal cancer. Methods: A total of 120 cases of sporadic dMMR colorectal cancer from July 2015 to April 2021 were retrospectively collected in Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College. Patients with Lynch syndrome; synchronous multiple colorectal cancers; preoperative anti-tumor treatments such as chemotherapy and radiotherapy; and those with incomplete follow-up information were excluded based on family history and next-generation sequencing (NGS) test results. Immunohistochemical stains were used to detect the expression of mismatch repair proteins, methylation-specific PCR for methylation testing, and fluorescent PCR for BRAF V600E gene mutation detection. The clinical and pathological data, and gene mutation status were analyzed. Follow-up was done to assess survival and prognosis including progression-free survival and overall survival rate. Results: Sporadic dMMR colorectal cancer occurred more frequently in the right side of the colon, in females, and in the elderly. Morphologically, it was mostly moderately-differentiated, and most patients had low-grade tumor budding. In terms of immunohistochemical expression, MLH1 and PMS2 loss were dominant, and there were age and location-specificities in protein expression. MLH1 methylation was commonly detected in elderly female patients and rare in young male patients; while MLH1 and PMS2 deficiency, and BRAF V600E mutation occurred more often on the right side (P<0.05). The 3-year and 5-year progression-free survival rates were 90.7% and 88.7% respectively, and the 3-year and 5-year overall survival rates were 92.8% and 90.7% respectively. Tumor budding status was an independent risk factor affecting patient recurrence (hazard ratio=3.375, 95% confidence interval: 1.060-10.741, P=0.039), patients with low-grade tumor budding had better prognosis, and those with medium or high-grade tumor budding had poor prognosis. Conclusion: For dMMR colorectal cancer patients, tumor budding status is an independent risk factor for recurrence.

ARTN-GFRA3 axis induces epithelial-mesenchymal transition phenotypes, migration, and invasion of gastric cancer cells via KRAS signaling.

Neural invasion underlies the local spread of gastric cancer and is associated with poor prognosis. This process has been receiving increasing attention in recent years. However, the relationship between neural invasion and the malignant phenotypes of gastric cancer cells, as well as the molecular mechanism involved in this process, remain unclear. In this study, bioinformatics analysis was performed using a dataset obtained from The Cancer Genome Atlas-Stomach Adenocarcinoma. The results revealed that high expression of GDNF family receptor alpha 3 (GFRA3) was associated with a poor prognosis of patients with gastric cancer. GFRA3 is a receptor for artemin (ARTN), a glial cell line-derived neurotrophic factor (GDNF). This association was indicated by short overall/disease-free survival, as well as the presence of high-stage and high-grade disease. Gene set enrichment analysis showed that two cancer-associated pathways, namely KRAS signaling and epithelial-mesenchymal transition (EMT), were activated when GFRA3 was highly expressed in gastric cancer. Further studies confirmed that GFRA3 activated KRAS downstream signaling phosphatidylinositol 3 kinase/protein kinase B (PI3K/AKT) or extracellular signal-regulated kinase (ERK) and induced EMT markers, as well as promoted the migration and invasion of gastric cancer cells. As a ligand of GFRA3, ARTN induced the EMT, migration, and invasion of gastric cancer cells via GFRA3. Notably, the effects of the ARTN-GFRA3 axis were attenuated by treatment with a KRAS inhibitor. The present findings indicated that, during the neural invasion of gastric cancer, ARTN-mediated activation of GFRA3 induces EMT phenotypes, migration, and invasion of gastric cancer cells via KRAS signaling.

[Effects of polyphyllin â ¦ on proliferation, apoptosis and cell cycle of diffuse large B-cell lymphoma cells].

The aim of this study was to investigate the effects of polyphyllin Ⅶ (PP Ⅶ) on proliferation, apoptosis, and cell cycle of diffuse large B-cell lymphoma (PLBCL) cell lines U2932 and SUDHL-4. The DLBCL cell lines were divided into a control group and a PPⅦ group, and experiments were conducted using MTT assay, flow cytometry, and Western blotting.Results showed that compared with the control group, PPⅦ significantly inhibited the proliferation of U2932 and SUDHL-4 cells (P<0.05). Apoptosis assays demonstrated that treatment with 0.50 and 1.00 µmol/L PP Ⅶ significantly increased the apoptosis rates of both cell lines (P<0.05), upregulated apoptosis-related proteins, and downregulated Bcl-2 protein level (P<0.05). Cell cycle analysis revealed that PPⅦ treatment led to an increase in G0/G1-phase cells (P<0.05) and a decrease in G2/M-phase cells (P<0.05), significantly downregulated cyclin D1, CDK4, CDK6, and survivin protein expression (P<0.05). In conclusion, PPⅦ exerted anti-lymphoma effects by inhibiting proliferation, promoting apoptosis, and inducing G0/G1 phase arrest in DLBCL cells.

Complete response of metastatic microsatellite-stable BRAF V600E colorectal cancer to first-line oxaliplatin-based chemotherapy and immune checkpoint blockade.

The randomized METIMMOX trial (NCT03388190) examined if patients with previously untreated, unresectable abdominal metastases from microsatellite-stable (MSS) colorectal cancer (CRC) might benefit from potentially immunogenic, short-course oxaliplatin-based chemotherapy alternating with immune checkpoint blockade (ICB). Three of 38 patients assigned to this experimental treatment had metastases from BRAF-mutant MSS-CRC, in general a poor-prognostic subgroup explored here. The ≥70-year-old females presented with ascending colon adenocarcinomas with intermediate tumor mutational burden (6.2-11.8 mutations per megabase). All experienced early disappearance of the primary tumor followed by complete response of all overt metastatic disease, resulting in progression-free survival as long as 20-35 months. However, they encountered recurrence at previously unaffected sites and ultimately sanctuary organs, or as intrahepatic tumor evolution reflected in the terminal loss of initially induced T-cell clonality in liver metastases. Yet, the remarkable first-line responses to short-course oxaliplatin-based chemotherapy alternating with ICB may offer a novel therapeutic option to a particularly hard-to-treat MSS-CRC subgroup.

Joint DNA-RNA-based NGS for diagnosis and treatment of a rare CD47-MET fusion lung adenocarcinoma which was immunoresistant and savoltinib-sensitive: a case report.

Targeted therapy and immunotherapy are both important in the treatment of non-small-cell lung cancer (NSCLC). Accurate diagnose and precise treatment are key in achieving long survival of patients. MET fusion is a rare oncogenic factor, whose optimal detection and treatment are not well established. Here, we report on a 32-year-old female lung adenocarcinoma patient with positive PD-L1 and negative driver gene detected by DNA-based next-generation sequencing (NGS). A radical resection of the primary lesion after chemotherapy combined with PD-1 checkpoint inhibitor administration indicated primary immuno-resistance according to her pathological response and rapid relapse. A rare CD47-MET was detected by RNA-based NGS, which was confirmed by fluorescence in situ hybridization. Multiplex immunofluorescence revealed a PD-L1 related heterogeneous immunosuppressive microenvironment with little distribution of CD4+ T cells and CD8+ T cells. Savolitinib therapy resulted in a progression-free survival (PFS) of >12 months, until a new secondary resistance mutation in MET p.D1228H was detected by re-biopsy and joint DNA-RNA-based NGS after disease progression. In this case, CD47-MET fusion NSCLC was primarily resistant to immunotherapy, sensitive to savolitinib, and developed secondary MET p.D1228H mutation after targeted treatment. DNA-RNA-based NGS is useful in the detection of such molecular events and tracking of secondary mutations in drug resistance. To this end, DNA-RNA-based NGS may be of better value in guiding precise diagnosis and individualized treatment in this patient population.

Exosome-shuttled miR-150-5p from LPS-preconditioned mesenchymal stem cells down-regulate PI3K/Akt/mTOR pathway via Irs1 to enhance M2 macrophage polarization and confer protection against sepsis.

Rationale: Sepsis is a life-threatening organ dysfunction and lack of effective measures in the current. Exosomes from mesenchymal stem cells (MSCs) reported to alleviate inflammation during sepsis, and the preconditioning of MSCs could enhance their paracrine potential. Therefore, this study investigated whether exosomes secreted by lipopolysaccharide (LPS)-pretreated MSCs exert superior antiseptic effects, and explored the underlying molecular mechanisms. Methods: Exosomes were isolated and characterized from the supernatants of MSCs. The therapeutic efficacy of normal exosomes (Exo) and LPS-pretreated exosomes (LPS-Exo) were evaluated in terms of survival rates, inflammatory response, and organ damage in an LPS-induced sepsis model. Macrophages were stimulated with LPS and treated with Exo or LPS-Exo to confirm the results of the in vivo studies, and to explain the potential mechanisms. Results: LPS-Exo were shown to inhibit aberrant pro-inflammatory cytokines, prevent organ damages, and improve survival rates of the septic mice to a greater extent than Exo. In vitro, LPS-Exo significantly promoted the M2 polarization of macrophages exposed to inflammation. miRNA sequencing and qRT-PCR analysis identified the remarkable expression of miR-150-5p in LPS-Exo compared to that in Exo, and exosomal miR-150-5p was transferred into recipient macrophages and mediated macrophage polarization. Further investigation demonstrated that miR-150-5p targets Irs1 in recipient macrophages and subsequently modulates macrophage plasticity by down-regulating the PI3K/Akt/mTOR pathway. Conclusion: The current findings highly suggest that exosomes derived from LPS pre-conditioned MSCs represent a promising cell-free therapeutic method and highlight miR-150-5p as a novel molecular target for regulating immune hyperactivation during sepsis.

Selective PI3Kδ inhibitor TYM-3-98 suppresses AKT/mTOR/SREBP1-mediated lipogenesis and promotes ferroptosis in KRAS-mutant colorectal cancer.

Colorectal cancer (CRC) is one of the most common tumors of the digestive system worldwide. KRAS mutations limit the use of anti-EGFR antibodies in combination with chemotherapy for the treatment of CRC. Therefore, novel targeted therapies are needed to overcome the KRAS-induced oncogenesis. Recent evidence suggests that inhibition of PI3K led to ferroptosis, a nonapoptotic cell death closely related to KRAS-mutant cells. Here, we showed that a selective PI3Kδ inhibitor TYM-3-98 can suppress the AKT/mTOR signaling and activate the ferroptosis pathway in KRAS-mutant CRC cells in a concentration-dependent manner. This was evidenced by the lipid peroxidation, iron accumulation, and depletion of GSH. Moreover, the overexpression of the sterol regulatory element-binding protein 1 (SREBP1), a downstream transcription factor regulating lipid metabolism, conferred CRC cells greater resistance to ferroptosis induced by TYM-3-98. In addition, the effect of TYM-3-98 was confirmed in a xenograft mouse model, which demonstrated significant tumor suppression without obvious hepatoxicity or renal toxicity. Taken together, our work demonstrated that the induction of ferroptosis contributed to the PI3Kδ inhibitor-induced cell death via the suppression of AKT/mTOR/SREBP1-mediated lipogenesis, thus displaying a promising therapeutic effect of TYM-3-98 in CRC treatment.

Zona incerta mediates early life isoflurane-induced fear memory deficits.

The potential long-term effects of anesthesia on cognitive development, especially in neonates and infants, have raised concerns. However, our understanding of its underlying mechanisms and effective treatments is still limited. In this study, we found that early exposure to isoflurane (ISO) impaired fear memory retrieval, which was reversed by dexmedetomidine (DEX) pre-treatment. Measurement of c-fos expression revealed that ISO exposure significantly increased neuronal activation in the zona incerta (ZI). Fiber photometry recording showed that ZI neurons from ISO mice displayed enhanced calcium activity during retrieval of fear memory compared to the control group, while DEX treatment reduced this enhanced calcium activity. Chemogenetic inhibition of ZI neurons effectively rescued the impairments caused by ISO exposure. These findings suggest that the ZI may play a pivotal role in mediating the cognitive effects of anesthetics, offering a potential therapeutic target for preventing anesthesia-related cognitive impairments.

Melatonin regulates mitochondrial function to alleviate ferroptosis through the MT2/Akt signaling pathway in swine testicular cells.

Increasing evidence has shown that many environmental and toxic factors can cause testicular damage, leading to testicular ferroptosis and subsequent male reproductive disorders. Melatonin is a major hormone and plays an vital role in regulating male reproduction. However, there is a lack of research on whether Mel can alleviate testicular cell ferroptosis and its specific mechanism. In this study, the results indicated that Mel could enhance the viability of swine testis cells undergoing ferroptosis, reduce LDH enzyme release, increase mitochondrial membrane potential, and affect the expression of ferroptosis biomarkers. Furthermore, we found that melatonin depended on melatonin receptor 1B to exert these functions. Detection of MMP and ferroptosis biomarker protein expression confirmed that MT2 acted through the downstream Akt signaling pathway. Moreover, inhibition of the Akt signaling pathway can eliminate the protective effect of melatonin on ferroptosis, inhibit AMPK phosphorylation, reduce the expression of mitochondrial gated channel (VDAC2/3), and affect mitochondrial DNA transcription and ATP content. These results suggest that melatonin exerts a beneficial effect on mitochondrial function to mitigate ferroptosis through the MT2/Akt signaling pathway in ST cells.

LPS exacerbates TRPV4-mediated itch through the intracellular TLR4-PI3K signalling.

Pruritus is often accompanied with bacterial infections, but the underlying mechanism is not fully understood. Although previous studies revealed that lipopolysaccharides (LPS) could directly activate TRPV4 channel and TRPV4 is involved in the generation of both acute itch and chronic itch, whether and how LPS affects TRPV4-mediated itch sensation remains unclear. Here, we showed that LPS-mediated TRPV4 sensitization exacerbated GSK101-induced scratching behaviour in mice. Moreover, this effect was compromised in TLR4-knockout mice, suggesting LPS acted through a TLR4-dependent mechanism. Mechanistically, LPS enhanced GSK101-evoked calcium influx in mouse ear skin cells and HEK293T cells transfected with TRPV4. Further, LPS sensitized TRPV4 channel through the intracellular TLR4-PI3K-AKT signalling. In summary, our study found a modulatory role of LPS in TRPV4 function and highlighted the TLR4-TRPV4 interaction in itch signal amplification.

Chemoprotective effect of arbutin on azoxymethane-induced aberrant crypt foci in rat colon via modulation of PCNA/Bax protein.

Arbutin is utilized in traditional remedies to cure numerous syndromes because of its anti-microbial, antioxidant, and anti-inflammatory properties. This study aimed to evaluate chemopreventive effects of arbutin on azoxymethane (AOM)-induced colon aberrant crypt foci (ACF) in rats. Five groups of rats were used: normal control group (rats injected hypodermically with sterile phosphate-buffered saline once per week for two weeks) and groups 2-5, which were subcutaneously inoculated with 15 mg/kg AOM once a week for two weeks. AOM control and 5-fluorouracil (5-FU) control groups were fed 10% Tween orally daily for 8 weeks using a feeding tube. The treated groups were fed 30 and 60 mg/kg arbutin every day for 2 months. ACF from the AOM control group had aberrant nuclei in addition to multilayered cells and an absence of goblet cells. The negative control group displayed spherical cells and nuclei in basal positions. Histological examination revealed a reduced number of AFC cells from colon tissues of the 5-FU reference group. Arbutin-fed animals showed down-regulation of proliferating cell nuclear antigen (PCNA) and up-regulation of Bax protein compared to AOM control. Rats fed with arbutin displayed a significant increase of superoxide dismutase (SOD) and catalase (CAT) activities in colon tissue homogenates compared to the AOM control group. In conclusion, arbutin showed therapeutic effects against colorectal cancer, explained by its ability to significantly decrease ACF, down-regulate PCNA protein, and up-regulate Bax protein. In addition, arbutin significantly increased SOD and CAT, and decreased malondialdehyde (MDA) levels, which might be due to its anti-proliferative and antioxidant properties.

Protective effect of Sasa veitchii extract against all-trans-retinoic acid-induced inhibition of proliferation of cultured human palate cells.

Cleft palate is the most common facial birth defect worldwide. It is caused by environmental factors or genetic mutations. Environmental factors such as pharmaceutical exposure in women are known to induce cleft palate. The aim of the present study was to investigate the protective effect of Sasa veitchii extract against medicine-induced inhibition of proliferation of human embryonic palatal mesenchymal cells. We demonstrated that all-trans-retinoic acid inhibited human embryonic palatal mesenchymal cell proliferation in a dose-dependent manner, whereas dexamethasone treatment had no effect on cell proliferation. Cotreatment with Sasa veitchii extract repressed all-trans-retinoic acid-induced toxicity in human embryonic palatal mesenchymal cells. We found that cotreatment with Sasa veitchii extract protected all-trans-retinoic acid-induced cyclin D1 downregulation in human embryonic palatal mesenchymal cells. Furthermore, Sasa veitchii extract suppressed all-trans-retinoic acid-induced miR-4680-3p expression. Additionally, the expression levels of the genes that function downstream of the target genes ( ERBB2 and JADE1 ) of miR-4680-3p in signaling pathways were enhanced by cotreatment with Sasa veitchii extract and all-trans-retinoic acid compared to all-trans-retinoic acid treatment. These results suggest that Sasa veitchii extract suppresses all-trans-retinoic acid-induced inhibition of cell proliferation via modulation of miR-4680-3p expression.

[Role of CTGF and PI3K/Akt signaling pathway in paraquat-induced mesenchymal changes in alveolar epithelial cells].

Objective: To investigate the role of connective tissue growth factor (CTGF) and PI3K/Akt signaling pathways in paraquat (PQ) -induced alterations in alveolar epithelial cell mesenchymalization (EMT) . Methods: In February 2023, RLE-6TN cells were divided into 2 groups, which were set as uncontaminated group and contaminated group (200 µmol/L PQ), and cellular EMT alteration, CTGF and PI3K/Akt signaling pathway related molecules expression were detected by cell scratch assay, qRT-PCR and western-blot assay. Using shRNA interference technology to specifically inhibit the expression of CTGF, RLE-6TN cells were divided into four groups: control group, PQ group (200 µmol/L PQ), interference group (transfected with a plasmid with shRNA-CTGF+200 µmol/L PQ), and null-loaded group (transfected with a plasmid with scramble- CTGF+200 µmol/L PQ), qRT-PCR and western blot were used to examine the alteration of the cellular EMT and the expression of molecules related to the activity of PI3K/Akt pathway. The PI3K/Akt signaling pathway was blocked by the PI3K inhibitor LY294002, and the expression of EMT-related molecules in cells of the control group, PQ group (200 µmol/L PQ), and inhibitor group (200 µmol/L PQ+20 µmol/L LY294002) was examined by qRT-PCR and western blot.The t-test was used to compare the differences between the two groups, while the analysis of variance (ANOVA) was applied to compare the differences among multiple groups. For further pairwise comparisons, the Bonferroni method was adopted. Results: The results of cell scratch test showed that compared with the uncontaminated group, RLE-6TN cells in the contaminated group had faster migration rate, lower mRNA and protein expression levels of E-Cadherin, and higher mRNA and protein expression levels of α-SMA, CTGF, PI3K and Akt, with statistical significance (P<0.05). After specific inhibition of CTGF expression, the mRNA and protein expression of CTGF, PI3K, Akt, and α-SMA in the cells of the interference group were significantly lower than that of the PQ group and the null-loaded group (P<0.05/6), whereas that of E-Cadherin was higher than that of the PQ group and the null-loaded group (P<0.05/6). Specifically blocking the PI3K/Akt signaling pathway, the mRNA and protein expression of PI3K, Akt and α-SMA in the cells of the inhibitor group was decreased compared with that of the PQ group (P<0.05/3), while the expression of E-Cadherin was elevated compared with that of the PQ group (P<0.05/3) . Conclusion: CTGF may promote PQ-induced alveolar epithelial cell EMT through activation of the PI3K/Akt signaling pathway. Inhibition of CTGF expression or blockade of PI3K/Akt signaling pathway activity can alleviate the extent of PQ-induced alveolar epithelial cell EMT.

A novel variant in the FLCN gene in a Chinese family with Birt-Hogg-Dubé syndrome.

BACKGROUND: This study aimed to identify disease-causing variants within a Chinese family affected by Birt-Hogg-Dubé syndrome (BHDS), which arises from an autosomal dominant inheritance pattern attributed to variants in the folliculin (FLCN) gene, recognized as a tumor suppressor gene. METHODS: A Chinese proband diagnosed with BHDS due to renal tumors underwent next-generation sequencing (NGS), revealing a novel variant in the FLCN gene. Sanger sequencing was subsequently performed on blood samples obtained from family members to confirm the presence of this variant. RESULTS: A novel germline frameshift variant (NM_144997.5:c.977dup) was identified in five individuals among the screened family members, marking the first report of this variant. Additionally, a somatic frameshift variant (NM_144997.5:c.1252del) was detected in the renal tumors of the proband. No variant was detected in unaffected family members. CONCLUSIONS: A novel heterozygous variant was identified in exon 9 of the FLCN gene, which broadens the spectrum of FLCN variants. We recommend that molecular analysis of the FLCN gene be performed in patients with suspected BHDS and their families.

Diospyros rhodocalyx Kurz induces mitochondrial-mediated apoptosis via BAX, Bcl-2, and caspase-3 pathways in LNCaP human prostate cancer cell line.

Background: Prostate cancer (PCa) is one of the causes of death in men worldwide. Although treatment strategies have been developed, the recurrence of the disease and consequential side effects remain an essential concern. Diospyros rhodocalyx Kurz, a traditional Thai medicine, exhibits diverse therapeutic properties, including anti-cancer activity. However, its anti-cancer activity against prostate cancer has not been thoroughly explored. This study aims to evaluate the anti-cancer activity and underlying mechanisms of the ethyl acetate extract of D. rhodocalyx Kurz (EADR) related to apoptosis induction in the LNCaP human prostate cancer cell line. Methods: Ethyl acetate was employed to extract the dried bark of D. rhodocalyx Kurz. The cytotoxicity of EADR on both LNCaP and WPMY-1 cells (normal human prostatic myofibroblast cell line) was evaluated using MTS assay. The effect of EADR on the cell cycle, apoptosis induction, and alteration in mitochondrial membrane potential (MMP) was assessed by the staining with propidium iodide (PI), Annexin V-FITC/PI, and JC-1 dye, respectively. Subsequent analysis was conducted using flow cytometry. The expression of cleaved caspase-3, BAX, and Bcl-2 was examined by Western blotting. The phytochemical profiling of the EADR was performed using gas chromatography-mass spectrometry (GC-MS). Results: EADR exhibited a dose-dependent manner cytotoxic effect on LNCaP cells, with IC50 values of 15.43 and 12.35 µg/mL after 24 and 48 h, respectively. Although it also exhibited a cytotoxic effect on WPMY-1 cells, the effect was comparatively lower, with the IC50 values of 34.61 and 19.93 µg/mL after 24 and 48 h of exposure, respectively. Cell cycle analysis demonstrated that EADR did not induce cell cycle arrest in either LNCaP or WPMY-1 cells. However, it significantly increased the sub-G1 population in LNCaP cells, indicating a potential induction of apoptosis. The Annexin V-FITC/PI staining indicated that EADR significantly induced apoptosis in LNCaP cells. Subsequent investigation into the underlying mechanism of EADR-induced apoptosis revealed a reduction in MMP as evidenced by JC-1 staining. Moreover, Western blotting demonstrated that EADR treatment resulted in the upregulation of BAX, downregulation of BCL-2, and elevation of caspase-3 cleavage in LNCaP cells. Notably, the epilupeol was a prominent compound in EADR as identified by GC-MS. Conclusion: The EADR exhibits anti-cancer activity against the LNCaP human prostate cancer cell line by inducing cytotoxicity and apoptosis. Our findings suggest that EADR promotes apoptosis by upregulating pro-apoptotic BAX, whereas downregulation of anti-apoptotic Bcl-2 results in the reduction of MMP and the activation of caspase-3. Of particular interest is the presence of epilupeol, a major compound identified in EADR, which may hold promise as a candidate for the development of therapeutic agents for prostate cancer.

SP1 promotes high glucose-induced lens epithelial cell viability, migration and epithelial-mesenchymal transition via regulating FGF7 and PI3K/AKT pathway.

BACKGROUND: Diabetic cataract (DC) is a common complication of diabetes and its etiology and progression are multi-factorial. In this study, the roles of specific protein 1 (SP1) and fibroblast growth factor 7 (FGF7) in DC development were explored. METHODS: DC cell model was established by treating SRA01/04 cells with high glucose (HG). MTT assay was conducted to evaluate cell viability. Transwell assay and wound-healing assay were performed to assess cell migration and invasion. Western blot assay and qRT-PCR assay were conducted to measure the expression of N-cadherin, E-cadherin, Collagen I, Fibronectin, SP1 and FGF7 expression. CHIP assay and dual-luciferase reporter assay were conducted to analyze the combination between FGF7 and SP1. RESULTS: FGF7 was upregulated in DC patients and HG-induced SRA01/04 cells. HG treatment promoted SRA01/04 cell viability, migration, invasion and epithelial-mesenchymal transition (EMT), while FGF7 knockdown abated the effects. Transcription factor SP1 activated the transcription level of FGF7 and SP1 overexpression aggravated HG-induced SRA01/04 cell injury. SP1 silencing repressed HG-induced SRA01/04 cell viability, migration, invasion and EMT, but these effects were ameliorated by upregulating FGF7. Additionally, SP1 knockdown inhibited the PI3K/AKT pathway by regulating the transcription level of FGF7. CONCLUSION: Transcription factor SP1 activated the transcription level of FGF7 and the PI3K/AKT pathway to regulate HG-induced SRA01/04 cell viability, migration, invasion and EMT.

Polyamine and EIF5A hypusination downstream of c-Myc confers targeted therapy resistance in BRAF mutant melanoma.

BACKGROUND: BRAF inhibitors are widely employed in the treatment of melanoma with the BRAF V600E mutation. However, the development of resistance compromises their therapeutic efficacy. Diverse genomic and transcriptomic alterations are found in BRAF inhibitor resistant melanoma, posing a pressing need for convergent, druggable target that reverse therapy resistant tumor with different resistance mechanisms. METHODS: CRISPR-Cas9 screens were performed to identify novel target gene whose inhibition selectively targets A375VR, a BRAF V600E mutant cell line with acquired resistance to vemurafenib. Various in vitro and in vivo assays, including cell competition assay, water soluble tetrazolium (WST) assay, live-dead assay and xenograft assay were performed to confirm synergistic cell death. Liquid Chromatography-Mass Spectrometry analyses quantified polyamine biosynthesis and changes in proteome in vemurafenib resistant melanoma. EIF5A hypusination dependent protein translation and subsequent changes in mitochondrial biogenesis and activity were assayed by O-propargyl-puromycin labeling assay, mitotracker, mitoSOX labeling and seahorse assay. Bioinformatics analyses were used to identify the association of polyamine biosynthesis with BRAF inhibitor resistance and poor prognosis in melanoma patient cohorts. RESULTS: We elucidate the role of polyamine biosynthesis and its regulatory mechanisms in promoting BRAF inhibitor resistance. Leveraging CRISPR-Cas9 screens, we identify AMD1 (S-adenosylmethionine decarboxylase 1), a critical enzyme for polyamine biosynthesis, as a druggable target whose inhibition reduces vemurafenib resistance. Metabolomic and proteomic analyses reveal that polyamine biosynthesis is upregulated in vemurafenib-resistant cancer, resulting in enhanced EIF5A hypusination, translation of mitochondrial proteins and oxidative phosphorylation. We also identify that sustained c-Myc levels in vemurafenib-resistant cancer are responsible for elevated polyamine biosynthesis. Inhibition of polyamine biosynthesis or c-Myc reversed vemurafenib resistance both in vitro cell line models and in vivo in a xenograft model. Polyamine biosynthesis signature is associated with poor prognosis and shorter progression free survival after BRAF/MAPK inhibitor treatment in melanoma cohorts, highlighting the clinical relevance of our findings. CONCLUSIONS: Our findings delineate the molecular mechanisms involving polyamine-EIF5A hypusination-mitochondrial respiration pathway conferring BRAF inhibitor resistance in melanoma. These targets will serve as effective therapeutic targets that can maximize the therapeutic efficacy of existing BRAF inhibitors.