Synergistic effects of the combination of trametinib and alpelisib in anaplastic thyroid cancer with BRAF and PI3KCA co-mutations.

Background: Anaplastic thyroid cancer (ATC), a rare and aggressive malignancy with a poor prognosis, has shown promise with the approved dabrafenib/trametinib combination for BRAFV600E mutation. Co-occurring PI3KCA mutations, identified as negative prognostic factors in lung cancer with BRAFV600E mutation, emphasize the need to target both pathways. Exploring trametinib and alpelisib combination becomes crucial for ATC. Methods: A patient-derived xenograft (PDX) and primary cell line were obtained from an ATC patient with BRAF and PI3KCA co-mutation. Individual testing of targeted therapies against BRAF, MEK, and PI3KCA was followed by a combination treatment. Synergistic effects were evaluated using the combination index. Immunoblotting assessed the efficacy, with validation performed using a PDX model. Results: In this study, the ATC0802 cell line and PDX were established from a refractory ATC patient. NGS revealed BRAF and PI3KCA co-mutations pre- and post-dabrafenib/trametinib treatment. Trametinib/alpelisib combination showed synergy, suppressing both pERK and pAKT levels, unlike monotherapies or BRAF knockdown. The combination induced apoptosis and, in the PDX model, demonstrated superior tumor growth inhibition compared to monotherapies. Conclusions: The combination of trametinib and alpelisib showed promise as a strategy for treating ATC with co-mutations in BRAF and PI3KCA, both in vitro and in vivo. This combination offers insights into overcoming resistance to BRAF-targeted treatments in ATC with mutations in BRAF and PI3KCA.

Wee1 inhibition by MK1775 potentiates gemcitabine through accumulated replication stress leading to apoptosis in biliary tract cancer.

Patients with advanced biliary tract cancer (BTC) have a poor prognosis, and novel treatments are needed. Gemcitabine, the standard of care for BTC, induces DNA damage; however, the ability of cancer cells to repair DNA dampens its effects. To improve the efficacy of gemcitabine, we combined it with MK1775, a Wee1 inhibitor that prevents activation of the G2/M checkpoint. BTC cell lines were treated with gemcitabine only or in combination with MK1775 to determine the therapeutic potential of BTC. Gemcitabine inhibited the growth and induced the apoptosis of four BTC cell lines to a greater extent when added with MK1775 than when added alone. The effects of the combination treatment were observed in both p53 wild-type and p53 mutant cell lines and were unaffected by knockdown of wild-type p53. The combination treatment increased the percentage of apoptotic cells and decreased the percentage of cells synthesizing DNA, suggesting that it caused DNA-damaged cells to accumulate and possibly die in S phase. It did not induce apoptosis when cells were arrested in mitosis using nocodazole. In a xenograft mouse model, gemcitabine plus MK1775 (but not either alone) inhibited the growth of tumors generated from inoculated BTC cells. Our results show that MK1775 highly enhances gemcitabine cytotoxicity in BTC regardless of p53 status. We suggest that the combination treatment elicits a DNA damage response and consequent apoptosis. Our preclinical study provides a basis for future clinical trials of gemcitabine plus MK1775 in patients with BTC.

Identification and Clinical Significance of Pancreatic Cancer Stem Cells and Their Chemotherapeutic Drug Resistance.

Pancreatic cancer ranks in the 10th-11th position among cancers affecting men in Taiwan, besides being a rather difficult-to-treat disease. The overall 5-year survival rate of pancreatic cancer is only 5-10%, while that of resectable pancreatic cancer is still approximately 15-20%. Cancer stem cells possess intrinsic detoxifying mechanisms that allow them to survive against conventional therapy by developing multidrug resistance. This study was conducted to investigate how to overcome chemoresistance and its mechanisms in pancreatic cancer stem cells (CSCs) using gemcitabine-resistant pancreatic cancer cell lines. Pancreatic CSCs were identified from human pancreatic cancer lines. To determine whether CSCs possess a chemoresistant phenotype, the sensitivity of unselected tumor cells, sorted CSCs, and tumor spheroid cells to fluorouracil (5-FU), gemcitabine (GEM), and cisplatin was analyzed under stem cell conditions or differentiating conditions. Although the mechanisms underlying multidrug resistance in CSCs are poorly understood, ABC transporters such as ABCG2, ABCB1, and ABCC1 are believed to be responsible. Therefore, we measured the mRNA expression levels of ABCG2, ABCB1, and ABCC1 by real-time RT-PCR. Our results showed that no significant differences were found in the effects of different concentrations of gemcitabine on CSCs CD44+/EpCAM+ of various PDAC cell line cultures (BxPC-3, Capan-1, and PANC-1). There was also no difference between CSCs and non-CSCs. Gemcitabine-resistant cells exhibited distinct morphological changes, including a spindle-shaped morphology, the appearance of pseudopodia, and reduced adhesion characteristics of transformed fibroblasts. These cells were found to be more invasive and migratory, and showed increased vimentin expression and decreased E-cadherin expression. Immunofluorescence and immunoblotting experiments demonstrated increased nuclear localization of total ß-catenin. These alterations are hallmarks of epithelial-to-mesenchymal transition (EMT). Resistant cells showed activation of the receptor protein tyrosine kinase c-Met and increased expression of the stem cell marker cluster of differentiation (CD) 24, CD44, and epithelial specific antigen (ESA). We concluded that the expression of the ABCG2 transporter protein was significantly higher in CD44+ and EpCAM+ CSCs of PDAC cell lines. Cancer stem-like cells exhibited chemoresistance. Gemcitabine-resistant pancreatic tumor cells were associated with EMT, a more aggressive and invasive phenotype of numerous solid tumors. Increased phosphorylation of c-Met may also be related to chemoresistance, and EMT and could be used as an attractive adjunctive chemotherapeutic target in pancreatic cancer.

Taiwan cobra envenoming: serum venom concentration before and after specific treatment and relationship with debridement of necrotic wound tissue.

Background: Bivalent freeze-dried neurotoxic (FN) antivenom has been the primary treatment since the 1980s for Taiwan cobra (Naja atra) envenomation in Taiwan. However, envenomation-related wound necrosis is a significant problem after cobra snakebites. In the present study, we analyzed the changes in serum venom concentration before and after antivenom administration to discover their clinical implications and the surgical treatment options for wound necrosis. Methods: The patients were divided into limb swelling and wound necrosis groups. The clinical outcome was that swelling started to subside 12 hours after antivenom treatment in the first group. Serum venom concentrations before and after using antivenoms were measured to assess the antivenom's ability to neutralize the circulating cobra venom. The venom levels in wound wet dressing gauzes, blister fluids, and debrided tissues were also investigated to determine their clinical significance. We also observed the evolutional changes of wound necrosis and chose a better wound debridement timing. Results: We prospectively enrolled 15 Taiwan cobra snakebite patients. Males accounted for most of this study population (n = 11, 73%). The wound necrosis group received more antivenom doses than the limb swelling group (4; IQR:2-6 vs 1; IQR:1-2, p = 0.05), and less records of serum venom concentrations changed before/after antivenom use (p = 0.0079). The necrotic wound site may release venom into circulation and cause more severe envenomation symptoms. Antivenom can efficiently diminish limb swelling in cobra bite patients. However, antivenom cannot reduce wound necrosis. Patients with early debridement of wound necrosis had a better limb outcome, while late or without debridement may have long-term hospital stay and distal limb morbidity. Conclusions: Antivenom can efficiently eliminate the circulating cobra venom in limb swelling patients without wound necrosis. Early debridement of the bite site wound and wet dressing management are suggestions for preventing extended tissue necrosis and hospital stay.

The Clinical Usefulness of Taiwan Bivalent Freeze-Dried Hemorrhagic Antivenom in Protobothrops mucrosquamatus- and Viridovipera stejnegeri-Envenomed Patients.

Snakebites from Protobothrops mucrosquamatus (Taiwan habus) and Viridovipera stejnegeri (green bamboo vipers) account for the most venomous snakebites in Taiwan. The bivalent freeze-dried hemorrhagic (FH) antivenom is employed to treat these two snakebite patients without a strict clinical trial. We evaluated the clinical usefulness of Taiwan bivalent freeze-dried hemorrhagic (FH) antivenom in Taiwan habu- and green bamboo viper-envenomed patients. We checked ELISA- based serum venom antigen levels before and after FH antivenom to evaluate FH's ability to neutralize patients' serum snake venom and its usefulness in reducing limb swelling after snakebites. Patients who had higher serum venom antigen levels had more severe limb swelling. Of the 33 enrolled patients, most of their snake venom antigen levels were undetected after the appliance of antivenom. Most enrolled patients (25/33) had their limb swelling subside within 12 h after antivenom treatment. The failure to reduce limb swelling was probably due to an inadequate antivenom dose applied in more severely envenomated patients. Our data indicate the feasibility of the FH antivenom in effectively eliminating venom and resolving the affected limb swelling caused by Taiwan habu and green bamboo viper bites.

Programming a nonvolatile memory-like sensor for KRAS gene sensing and signal enhancement.

A programmable field effect-based electrolyte-insulator-semiconductor (EIS) sensor constructed with a nonvolatile memory-like structure is proposed for KRAS gene DNA hybridization detection. This programmable EIS structure was fabricated with silicon oxide (SiO2)/silicon nitride (Si3N4)/silicon oxide on a p-type silicon wafer, namely electrolyte-oxide-nitride-oxide-Si (EONOS). In this research, voltage stress programming from 4 to 20V was applied to trigger holes confinement in the nitride-trapping layer that, consequently, enhances the DNA attachment onto the sensing surface due to additional electrostatic interaction. Not solely resulting from the higher DNA load, the programming may affect the orientation of the DNA that finally contributes to the change in capacitance. Findings have shown that a higher voltage program is able to increase the total capacitance and results in ~3.5- and ~5.5-times higher sensitivities for a series of concentrations for complementary DNA and wild type versus mutant DNA hybridization detection, respectively. Overall, it has been proven that the voltage program on the nonvolatile memory-like structure of EONOS is a notable candidate for genosensor development, scoping the diagnosis of a single nucleotide polymorphism (SNP)-related disease.

Expression of matrix metalloproteinases MMP-2 and MMP-9 in gastric cancer and their relation to claudin-4 expression.

Matrix metalloproteinases (MMPs) MMP-2 and MMP-9 can degrade type IV collagen of extracellular matrix and basal membranes. Claudin-4 is a member of a large family of transmembrane proteins, claudins, essential in the formation and maintenance of tight junctions. Claudin-4 has been shown to activate MMP-2, indicating that claudin-mediated increased cancer cell invasion might be mediated through the activation of MMP proteins. To explore the roles of MMP-2, MMP-9 and claudin-4 in gastric cancer, we selected 88 cases and then analyzed the expression of these proteins using immunohistochemistry. We found that all of MMP-2, MMP-9 and claudin-4 expressions were significantly higher in intestinal-type than in diffuse-type gastric cancer. On further analysis, testing the relationship between MMP-2 and MMP-9 expression with claudin-4 expression, claudin-4 expression was significantly associated with MMP-9 expression, but not with MMP-2 expression. The results showed that MMP-2, MMP-9 and claudin-4 expression may be phenotypic features, distinguishing intestinal-type and diffuse-type gastric cancer. Possibly, claudin-4 played a role in determining MMP-9 activity which favored intestinal-type gastric cancer to distal metastasis.

Differential expression of claudin-4 between intestinal and diffuse-type gastric cancer.

Our previous microarray analysis of gastric cancer found that claudin-4 was differentially expressed between intestinal-type gastric cancer (IGC) and diffuse-type gastric cancer (DGC). Claudin-4 is a member of a large family of transmembrane proteins, claudins, essential in the formation and maintenance of tight junctions. To explore the roles of claudin-4 in the two histologically distinct types of gastric cancer, we selected 45 IGC and 48 DGC cases and then analyzed the expression of the protein using immunohistochemistry. We found that the overexpression of claudin-4 was greater in IGC than in DGC. A trend was observed between the overexpression of claudin-4 and lymph node metastasis, however, this association was not statistically significant. The results showed that the expression of claudin-4 was lower in DGC. Possibly it played a role in determining the diffuse phenotype and loose cohesion of cells in DGC in a similar manner as E-cadherin.