Introduction: Colorectal cancer (CRC) is the third most commonly diagnosed cancer worldwide. Its incidence is influenced by genetics, age, lifestyle, and diet factors. Indeed, a balanced diet rich in fiber and regular physical activity can significantly reduce the risk of developing CRC. Functional Foods have aroused considerable interest due to their antiproliferative and anti-cancer effects. Interestingly, bergapten, the main furanocoumarin in bergamot, exhibiting antioxidant properties, has been particularly studied for its ability to inhibit proliferation and induce apoptosis in different tumour cell lines, including CRC. Paradoxically, several reports have highlighted the pro-oxidant effects of natural antioxidant compounds, which increase free radicals and exacerbate oxidative stress. Based on this evidence, we investigated the molecular mechanism by which bergapten may affect survival and ROS production in CRC cells.
Methods: CaCo-2 colorectal adenocarcinoma cells were used as a model to evaluate bergapten anti-cancer effects. ROS production and mitochondrial membrane potential were evaluated through flow cytometry. mRNA levels of stemness, the epithelial-to-mesenchymal transition (EMT), the cell cycle and apoptosis markers were quantified by qRT-PCR. Western blotting was carried out to detect protein levels.
Results: Low doses of bergapten induced a reduction in intracellular ROS levels in CaCo-2 cells, as is generally reported. Unexpectedly, high doses of the furanocumarin promoted a time-dependent significant increase in ROS levels. In CaCo-2 bergapten-treated cells, a reduction in mitochondrial membrane potential, a down-regulation of anti-apoptotic markers, and an increase in pro-apoptotic markers were observed, leading to cytochome C release. In agreement with this, bergapten induced a lowered expression of Cyclin D1, Notch family members, and Survivin compared to untreated cells. Interestingly, the bergapten-induced inhibition of Notch signalling also led to the down-regulation of the EMT, reducing CRC growth and progression.
Conclusions: These results suggest that bergapten, at high doses, may act as a promoter of oxidative stress, favoring antiproliferative effects through the activation of intrinsic apoptosis.

BACKGROUND AND AIM

The global incidence of melanoma is rising steadily. Despite significant advancements in therapeutic options, resistance mechanisms often emerge, leading to disease progression. This event underscores the importance of identifying novel intracellular targets. The transient receptor potential melastatin subtype 8 (TRPM8), a non-selective cation channel with a preference for calcium permeation, is aberrantly expressed in various solid malignancies, including melanoma. Thus, it could represent a promising therapeutic target in melanoma cells.

METHODS

Two melanoma cell lines, derived from subcutaneous and lymph node metastases, were utilized to investigate the effects of TRPM8 modulators. Multiple methodologies, including colorimetric assays, Western blotting, immunofluorescence (IF), and confocal microscopy, were employed to assess the biological impact of these modulators.

RESULTS

Time-course experiments demonstrated that TRPM8 modulators induced melanoma cell death. These findings were corroborated by dual-fluorescence IF analyses. Further investigation of the molecular mechanisms revealed that the apoptotic pathway triggered by these modulators involved caspase 3 activation.

CONCLUSIONS

Additional studies are necessary to elucidate the regulatory mechanisms of TRPM8 and the resulting calcium influx dynamics. Nonetheless, the identification of novel chemotherapeutic targets with enhanced potency and selectivity may pave the way for innovative melanoma treatment strategies. These approaches hold significant promise for advancing oncology therapeutics.

Background: The cyclin dependent kinase (CDK) 4/6 inhibitor palbociclib plus endocrine therapy is an effective treatment for patients with estrogen receptor (ER)-positive and HER2-negative breast cancer (BC). Notwithstanding promising clinical outcomes, intrinsic or acquired resistance frequently arises. Given the established role of cancer-associated fibroblasts (CAFs) in the resistance to chemotherapeutics and the implication of the G-protein estrogen coupled receptor (GPER) in mediating stromal functions, we sought to investigate whether CAFs may contribute to palbociclib resistance in BC cells via GPER.

Methods: MCF7 and T47D BC cells, as well as CAFs isolated from breast tumor tissues, were used as model systems. Docking experiments allowed us to evaluate the potential interaction of palbociclib with GPER. Immunoblotting, gene expression arrays, and gene silencing experiments along with 2D and 3D co-culture assays were performed to evaluate the activation of GPER signaling by palbociclib in CAFs. Investigations on large cohorts of BC patients served to assess new potential stromal targets involved in palbociclib resistance.

Results: First, docking studies showed that palbociclib can interact with the binding site of GPER, like its well-known ligands. Next, we found that in CAFs, palbociclib stimulates the main sensors of GPER activation like ERK1/2 activation and c‑Fos up-regulation. To assess the transcriptional changes mediated by palbociclib through GPER in CAFs, we performed a human chemokine gene expression array. Notably, we found that the palbociclib-induced increase of pro-inflammatory genes is no longer evident upon silencing GPER expression. Finally, GPER silencing in CAFs interfered with the capacity of palbociclib to reduce the viability of BC cells in co-culture assays.

Conclusions: Our findings suggest that the activation of GPER signaling in CAFs may contribute to palbociclib resistance in breast cancer cells. On this basis, therapeutic strategies targeting CAFs might enhance the efficacy of palbociclib in BC patients.

Introduction: Arteriovenous malformation of the brain (bAVM) is a vascular condition affecting brain arterioles. It most likely arises due to an impaired expression of vascular differentiation markers that leads failed capillary bed development and, then, to the arteriolar-to-venule direct shunt. In this context, EFNB2 and ephrin-B4 seem to drive arteriovenous differentiation. Although several pathways have been linked to disease development, knowledge on the molecular basis of this phenotype is still very limited.

Methods: Methylome analysis was performed in endothelial cells purified from bAVM in order to identify aberrant methylation patterns in genes controlling vascular development and endothelial cell/vascular smooth muscle cell (VSMC) cross-talk. Human cerebral microvascular endothelial cells (HCMECs) were used as the control. The results were validated by quantitative methylation-specific PCR and quantitative real-time PCR.

Results: Both CpG- and CHG-aberrant methylation events were identified in bAVM endothelial cells. Most differentially methylated loci included noncoding RNA genes. Genes already known to be linked to bAVM development were identified. Interestingly, we focused our attention on the EPHB1 gene, not yet linked to bAVM onset. Likewise, other differentially methylated genes included transcription factors expressed in VSMCs that regulate the expression of genes involved in endothelial cell differentiation.

Conclusion: Our data allow to identify aberrant methylation events occurring in bAVM endothelial cells when compared to HCMECs. Notably, these genes are clustered in pathways related to vascular homeostasis, as well as to VSMC-- endothelial cell crosstalk, suggesting that an impairment of this interaction plays a prominent role in the loss of vascular differentiation in bAVM phenotypes.

Poly(ADP-ribose) (PAR) glycohydrolase (PARG) serves as a key enzyme in the hydrolysis of PAR. Deficiency in PARG increases cell death upon exposure to γ-irradiation or treatment with an alkylating agent. This makes PARG a prospective therapeutic target for cancer. Our previous research illustrated that MO2455, leading to PAR accumulation, exerts cytotoxicity on B cell lymphoma cells by disrupting the B cell pathway. In this study, we explored and compared the cellular mechanisms underlying MO2455-induced cell death in histiocytic lymphoma U937, monocytic leukemia THP-1, and T lymphoblastic leukemia CCRF-CEM cells. MO2455 triggered cell death in all cell types at similar concentration ranges. Cell death was observed through the appearances of the apoptotic subG1 fraction and annexin-V+/propidium iodide (PI)- fraction, followed by the annexin-V+/PI+ fraction in U937 cells after MO2455 treatment. U937 cells showed a decreased mitochondrial membrane potential and an early increase in ɣ-H2AX levels 5 hrs after MO2455 treatment, suggesting a rapid DNA damage and apoptosis induction process. THP-1 also showed an increase in the apoptotic subG1 fraction and then the appearance of the annexin-V+/PI+ fraction. CCRF-CEM showed a rapid induction of the annexin-V+/PI+ fraction. Taken together, MO2455 can effectively induce cell death through some common and distinct processes in various types of lymphoma and leukemia cells.

Poly (ADP-ribose) polymerase (PARP) recognizes single- and double-strand DNA breaks and generates specific signals to facilitate DNA repair. In a genome-wide screening, we identified PARP-1 as a potential target for radiosensitization. PARP-1 is involved in the cellular response to DNA damage, and inhibiting its function can affect the repair process. Additionally, PARP-2, another member of the PARP family, also contributes to DNA repair and affects radiosensitivity. When both PARP-1 and PARP-2 are functionally inhibited, cells become more sensitive to ionizing radiation. In this study, we utilized several clinically approved PARP inhibitors to explore their effects on the radiosensitization in cancer cells. The combined impacts of γ-irradiation and PARP inhibitors on clonogenic cell survival, signal transduction, and cellular senescence were analyzed in cancer cells. We found that most of the PARP inhibitors showed radiosensitization effects on lung adenocarcinoma A549 cells. Rucaparib showed the highest enhancement ratio. Talazoparib showed radiosensitization at the lowest concentration. As a result of a combination of talazoparib and γ-irradiation, we observed an increased level of cellular senescence, accompanied by a decrease in the mitochondrial membrane potential. Our study provides insights into potential therapeutic strategies for enhancing cancer cell sensitivity to radiation. The effect of PARP inhibitors in combination with γ-irradiation on different types of cancer cell lines will also be discussed.

The analysis of the combined effects of anti-cancer drugs remains limited, especially with DNA quantity data analysis using flow cytometry (FCM). In this study, we aimed to develop a more applicable analytical method combining FCM and machine learning to evaluate the combined effects of anti-cancer drugs more objectively. Human gastric cancer cell lines MKN45, MKN28, and KATO III were treated with platinum-based drugs cisplatin (CDDP) and oxaliplatin (LOHP), both alone and in combination, for 24 hrs. Cells were then fixed and stained with propidium iodide (PI) for DNA content analysis by FCM. A random forest model using Python's scikit-learn was applied to the obtained dataset (comprising four groups: non-drug-treated control, CDDP alone, LOHP alone, and combination). The model successfully distinguished between groups with over 95% accuracy by utilizing not only PI fluorescence intensity but also cell size (with forward scatter, FSC) and internal structural complexity (with side scatter, SSC) as features. An analysis of the combination effects revealed different trends depending on the cell line, with MKN45 showing effects similar to LOHP alone and KATO III showing effects similar to CDDP alone. This novel analytical method enables a more objective evaluation of drug combination effects that are difficult to capture with conventional statistical analysis, potentially contributing to the optimization of future cancer treatment strategies.

The development of radiation sensitization methods for cancer cells with low toxicity to normal cells is considered to contribute to improving cancer radiotherapy. To date, a comprehensive approach using an shRNA library has been employed to identify candidate genes for γ-ray sensitization in cancer cells, with a focus on the BACE1 (β-site of amyloid precursor protein (APP)-cleaving enzyme 1) gene. The BACE1 gene mutation has been observed in lung cancer, bone tumors, ovarian cancer, and esophageal cancer. Meanwhile, the effect of inhibiting BACE2 on radiosensitivity, which functions similarly to BACE1 and is upregulated in cancer cells, has not been investigated.

It has previously been reported that the inhibition of BACE1 leads to an increase in γ-H2AX foci, a marker of DNA strand breaks, suggesting its involvement in the early response to DNA damage. To investigate this possibility, BACE1 was knocked down using siRNA in cancer cell lines, and their sensitivity to gamma-irradiation was examined. Also, using siRNA, we evaluated the radiosensitization effect of BACE2 inhibition on cancer cells, which show high BACE2 expression levels.

BACE1 knockdown showed an increased sensitivity to γ-ray irradiation in the cancer cell lines Trex-HeLa, MDA-MB-231, SAOS, U2OS, HeLa, and T98G, whereas BACE1 knockdown did not show a sensitization effect on the normal fibroblast cell line WI-38. BACE1 knockdown showed a greater radiosensitizing effect in HeLa and SAOS cells, which have dysfunctional p53, compared to U2OS cells with normal p53 function. BACE2 knockdown did not exert a radiosensitization effect in cancer cells. These results suggest that BACE1 may be a potential target for radiosensitization in certain cancer cells.

Introduction: E3 ubiquitin ligases (EUL) and de-ubiquitinases (DUB) that regulate DDR proteins may influence ovarian cancer pathogenesis. We evaluated DDR-regulating EULs and DUBs in ovarian cancers.

Methods: EULs (DDB2, CUL4A, HLTF, RAD18 and HUWE1) and DUBs (USP5, USP7, USP11 and PSMD14) were immunohistochemically profiled in 331 tumours, and the clinicopathological outcome were analysed. Transcriptomic analysis was completed on a publicly available data set (n=424).

Results: DDB2, USP7 and USP11 expression was nuclear. CUL4A, HLTF, RAD18, HUWE1, USP5 and PSMD14 showed both nuclear and cytoplasmic expression. EULs [low nuclear DDB2 and CUL4A cytoplasmic overexpression] and DUBs [USP5 (nuclear and cytoplasmic) overexpression] were associated with high-grade serous carcinoma (p = 0.02, 0.003, <0.001, 0.03, respectively). EULs [low DDB2 nuclear expression (p = 0.01) and CUL4A nuclear overexpression (p = 0.05)] and DUBs [USP5 cytoplasmic overexpression (p = 0.05) and PSMD14 cytoplasmic overexpression (p = 0.02)] were associated with advanced-stage tumours. In terms of survival outcomes, EULs [nuclear and cytoplasmic CUL4A (p = 0.05, <0.001, respectively), nuclear HLTF (p = 0.003) and low-nuclear DDB2 (p = 0.01)] and DUBs [overexpression of USP7 (p = 0.005), nuclear and cytoplasmic USP5 overexpression (p = 0.004, 0.04, respectively), cytoplasmic PSMD14 overexpression (p = 0.01), nuclear USP11 overexpression (p = 0.006)] were associated with poor progression-free survival (PFS). In a multivariate analysis, advanced stage, cytoplasmic CUL4A overexpression and nuclear USP5 overexpression remain independently associated with worse PFS (p = 0.02, 0.03, respectively). At the transcriptomic level, in p53 mutant advanced-stage tumours that received platinum-based chemotherapy (n=424), high USP5 remains associated with poor PFS.

Conclusion: Our data suggest a complex role of EULs and DUBs in ovarian cancers. USP5 may be an attractive target for patient stratification and therapeutics in ovarian cancer.

BACKGROUND-AIM

Colorectal cancer (CRC) is the third most common cancer and the second leading cause of cancer-related death worldwide. Epidemiological studies demonstrate that men have a higher CRC incidence rate and face worse survival outcomes compared to women. This gender disparity in CRC incidence and outcomes pushed us to explore the mechanisms by which the androgen receptor (AR) may influence its pathology. Interestingly, the roles of the AR in cancer extend beyond traditional hormone-dependent cancers; in fact, in recent years, researchers have identified that AR signaling is also implicated in the progression of other cancers that do not typically respond to hormonal regulation. Understanding how AR mediates CRC's aggressiveness could provide valuable insights that may help to find more effective treatment strategies.

METHODS

In this study, we used CRC-derived Caco2, LoVo, and HCT-116 cells, expressing the AR to different extents. A BrdU incorporation assay and the measurement of spheroid growth were used to monitor cell proliferation. Biochemical approaches such as co-immunoprecipitation, immunoblotting, and immunofluorescence show that androgen treatment induces an interaction between the AR and Filamin A and the activation of different effectors without altering AR cell localization.

RESULTS

Our findings demonstrate that, in CRC cells, androgen treatment triggers an interaction between the AR and Filamin A. This complex activates Rac, p70, PKCs, and other proteins, thereby controlling different CRC-derived cells' proliferation. The antiandrogen Bicalutamide and a small peptide, Rh2025u, designed to mimic the AR sequences responsible for the AR/Filamin A interaction, reverse the androgen-induced effects in all cell lines.

CONCLUSIONS

This study underscores the significance of the AR in CRC and suggests that variations in androgen levels may influence both the onset and progression of this disease. By elucidating the role of the AR in CRC, this research opens avenues for innovative screening strategies and the development of targeted therapies.