Polysaccharide extract of Spirulina sp. increases effector immune-cell killing activities against cholangiocarcinoma.

Cyanobacteria and algae serving as promising food supplements have recently garnered attention for their emerging potential in anti-cancer activity. Cholangiocarcinoma (CCA) or bile duct cancer is one of the top-leading cancers affecting people, particularly in Asian continent. With patients exhibiting no or minimal symptoms in the early stages, advanced CCA is often diagnosed, and primary treatments such as surgery may not be suitable. Discovery of natural bioactive compounds for cancer treatments have, thus, attracted attention as one of the effective means to combat CCA or to supplement primary treatments. In this work, ethanolic and polysaccharide extracts of cyanobacteria and algae were tested for their cytotoxicity against 2 CCA cell lines (KKU055 and KKU213A). The ethanolic extracts from Leptolyngbya sp. and Chlorella sp. demonstrated growth inhibition of both CCA cell lines, with IC50 values of 0.658 mg/mL and 0.687 mg/mL for KKU055, and 0.656 mg/mL and 0.450 mg/mL for KKU213A. In contrast, only the polysaccharide extracts from Sargassum spp. exhibited a remarkable cytotoxic effect, while the polysaccharide extract from Spirulina sp. showed slight effect only at a higher concentration (2 mg/mL). All tested extracts were further investigated for improving immune cell killing ability and showed that Spirulina sp. polysaccharide extract was able to improve the immune cell killing ability. This extract was then investigated for its effects on the immune cell population, which demonstrated to have positive impact on NK cell population. To further explore the potential use, synergistic effect of Spirulina sp. polysaccharide extract with an already-in-use chemotherapeutic drug, gemcitabine, on immune cell cytotoxicity was investigated. The results showed that the immune cell cytotoxicity was enhanced in the co-treatment compared to the use of each treatment separately. The most apparent difference was observed in KKU055 cells where % living cells were reduced from 78.96% (immune cell alone) to 20.93% when the combined gemcitabine and Spirulina sp. extracts were used.

Cordycepin exhibits both antiviral and anti-inflammatory effects against dengue virus infection.

Cordycepin, a natural derivative of adenosine from Cordyceps militaris, can inhibit the replication of the dengue virus (DENV). Here, we investigated its antiviral and anti-inflammatory effects in DENV infected cells. Cordycepin significantly inhibited DENV-2 infection, virion production, and viral protein synthesis. It also reduced DENV-induced cytokine/chemokine production, including RANTES, IP-10, IL-6, and TNF-α. Mechanistically, cordycepin targeted the DENV NS5 protein, suppressing RANTES expression and hindering viral replication. Additionally, it inhibited the NF-κB pathway, leading to reduced nuclear translocation and signaling deactivation. PCR array analysis revealed cordycepin's suppression of 46 genes associated with DENV-induced inflammation. These findings highlight cordycepin's dual potential as an antiviral and anti-inflammatory agent against DENV, making it as a promising candidate for dengue treatment, targeting both viral and host factors.

Enhancing cancer immunotherapy using cordycepin and Cordyceps militaris extract to sensitize cancer cells and modulate immune responses.

Integrating immunotherapy with natural compounds holds promise in enhancing the immune system's ability to eliminate cancer cells. Cordyceps militaris, a traditional Chinese medicine, emerges as a promising candidate in this regard. This study investigates the effects of cordycepin and C. militaris ethanolic extract (Cm-EE) on sensitizing cancer cells and regulating immune responses against breast cancer (BC) and hepatocellular carcinoma (HCC) cells. Cordycepin, pentostatin and adenosine were identified in Cm-EE. Cordycepin treatment decreased HLA-ABC-positive cells in pre-treated cancer cells, while Cm-EE increased NKG2D ligand and death receptor expression. Additionally, cordycepin enhanced NKG2D receptor and death ligand expression on CD3-negative effector immune cells, particularly on natural killer (NK) cells, while Cm-EE pre-treatment stimulated IL-2, IL-6, and IL-10 production. Co-culturing cancer cells with effector immune cells during cordycepin or Cm-EE incubation resulted in elevated cancer cell death. These findings highlight the potential of cordycepin and Cm-EE in improving the efficacy of cancer immunotherapy for BC and HCC.

Comprehensive phytochemical profiling and biological activities of Hodgsonia heteroclita subsp. indochinensis seed extracts.

Hodgsonia heteroclita subsp. indochinensis, a member of the Cucurbitaceae family, is utilized in traditional medicinal remedies based on indigenous wisdom. This study aimed to comprehensively identify and analyze the bioactive phytoconstituents within H. heteroclita subsp. indochinensis seeds. Seeds were sequentially extracted with n-hexane, ethyl acetate, and methanol. Liquid chromatography-mass spectrometry analysis detected ferulic acid, salicylic acid, cucurbitacin E, stigmasterol glucoside, and ß-sitosterol glucoside in all extracts. The total phenolic content in the HH(S)-EtOAc and HH(S)-MeOH was 14.22 ± 1.58 and 12.98 ± 1.03 mg gallic acid equivalent/g, respectively. Consequently, the HH(S)-EtOAc demonstrated antioxidant activity with an IC50 of 1.10 ± 0.28 mg/mL, while the HH(S)-MeOH displayed strong antioxidant potential with an IC50 of 0.04 ± 0.00 mg/mL according to an ABTS assay. Antibacterial evaluations of both the HH(S)-hexane and HH(S)-EtOAc revealed significant activity against Staphylococcus aureus (zone of inhibition (ZOI): 13.67 ± 2.31 and 11.67 ± 1.53 mm, respectively) but limited activity against Escherichia coli (ZOI: 7.33 ± 0.58 and 7.67 ± 0.58 mm, respectively). Additionally, the extracts exhibited low minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values, ranging from 62.50 to 250 mg/mL. The antiproliferative activity of seed extracts was assessed against two breast cancer cell lines (MCF-7 and MDA-MB-231), normal breast cells (MCF10A), and human embryonic kidney (HEK) 293T cells, through MTT and clonogenic assays. The results revealed IC50 values exceeding 400 µg/mL, indicating that the extracts are safe. Furthermore, all seed extracts (50 µg/mL) exhibited potent anti-inflammatory activity, evident by their substantial inhibition of nitric oxide production (p < 0.001) and inducible nitric oxide synthase (iNOS) gene expression (p < 0.05) in LPS-induced RAW264.7. These findings demonstrate the potential for H. heteroclita subsp. indochinensis seed extracts in the development of functional foods, nutraceuticals, and dietary supplements due to their diverse bioactive compounds and substantial biological activities, particularly their anti-inflammatory effects.

Inhibition of Oral Pathogenic Bacteria, Suppression of Bacterial Adhesion and Invasion on Human Squamous Carcinoma Cell Line (HSC-4 Cells), and Antioxidant Activity of Plant Extracts from Acanthaceae Family.

Medicinal plants have traditionally been used to treat various human diseases worldwide. In this study, we evaluated the leaf extracts of plants from the Acanthaceae family, specifically Clinacanthus nutans (Burm.f.) Lindau, Thunbergia laurifolia Lindl., and Acanthus ebracteatus Vahl., for their compounds and antioxidant activity. The ethanolic extracts of A. ebracteatus showed the highest total phenolic content at 22.55 mg GAE/g extract and the strongest antioxidant activities, with IC50 values of 0.24 mg/mL and 3.05 mg/mL, as determined by DPPH and ABTS assays. The antibacterial efficacy of these extracts was also tested against Streptococcus pyogenes, Streptococcus mutans, Staphylococcus aureus, and Klebsiella pneumoniae. The diameters of the inhibition zones ranged from 14.7 to 17.3 mm using the agar well diffusion method, with MIC and MBC values ranging from 7.81 to 250 mg/mL. Anti-biofilm formation, antibacterial adhesion, and antibacterial invasion assays further demonstrated that these medicinal plant extracts can inhibit bacterial biofilm formation and prevent the adhesion and invasion of oral pathogenic bacteria on the human tongue squamous cell carcinoma-derived cell line (HSC-4 cells). The ethanolic extracts of C. nutans and A. ebracteatus were able to inhibit the gtfD and gbp genes, which facilitate biofilm formation and bacterial adherence to surfaces. These findings provide new insights into the antibacterial and antioxidant properties of plant extracts from the Acanthaceae family. These activities could enhance the clinical and pharmaceutical applications of plant extracts as an alternative therapy for bacterial infections and a dietary supplement.

Clinacanthus nutans (Burm. f.) Lindau Extract Inhibits Dengue Virus Infection and Inflammation in the Huh7 Hepatoma Cell Line.

Dengue virus (DENV) infection has emerged as a global health problem, with no specific treatment available presently. Clinacanthus nutans (Burm. f.) Lindau extract has been used in traditional medicine for its anti-inflammatory and antiviral properties. We thus hypothesized C. nutans had a broad-ranged activity to inhibit DENV and the liver inflammation caused by DENV infection. The study showed that treatment using C. nutans extract during DENV infection (co-infection step) showed the highest efficiency in lowering the viral antigen concentration to 22.87 ± 6.49% at 31.25 µg/mL. In addition, the virus-host cell binding assay demonstrated that C. nutans treatment greatly inhibited the virus after its binding to Huh7 cells. Moreover, it could remarkably lower the expression of cytokine and chemokine genes, including TNF-α, CXCL10, IL-6, and IL-8, in addition to inflammatory mediator COX-2 genes. Interestingly, the activation of the NF-κB signaling cascade after C. nutans extract treatment was dramatically decreased, which could be the underlying mechanism of its anti-inflammatory activity. The HPLC profile showed that gallic acid was the bioactive compound of C. nutans extract and might be responsible for the antiviral properties of C. nutans. Taken together, our results revealed the potential of C. nutans extract to inhibit DENV infection and lower inflammation in infected cells.

Tri-specific killer engager: unleashing multi-synergic power against cancer.

Cancer continues to be a global health concern, necessitating innovative solutions for treatment. Tri-specific killer engagers (TriKEs) have emerged as a promising class of immunotherapeutic agents, offering a multifaceted approach to cancer treatment. TriKEs simultaneously engage and activate natural killer (NK) cells while specifically targeting cancer cells, representing an outstanding advancement in immunotherapy. This review explores the generation and mechanisms of TriKEs, highlighting their advantages over other immunotherapies and discussing their potential impact on clinical trials and cancer treatment. TriKEs are composed of three distinct domains, primarily antibody-derived building blocks, linked together by short amino acid sequences. They incorporate critical elements, anti-cluster of differentiation 16 (CD16) and interleukin-15 (IL-15), which activate and enhance NK cell function, together with specific antibody to target each cancer. TriKEs exhibit remarkable potential in preclinical and early clinical studies across various cancer types, making them a versatile tool in cancer immunotherapy. Comparative analyses with other immunotherapies, such as chimeric antigen receptor-T (CAR-T) cell therapy, immune checkpoint inhibitors (ICIs), cytokine therapies, and monoclonal antibodies (mAbs), reveal the unique advantages of TriKEs. They offer a safer pathway for immunotherapy by targeting cancer cells without hyperactivating T cells, reducing off-target effects and complications. The future of TriKEs involves addressing challenges related to dosing, tumor-associated antigen (TAA) expression, and NK cell suppression. Researchers are exploring innovative dosing strategies, enhancing specificity through tumor-specific antigens (TSAs), and combining TriKEs with other therapies for increased efficacy.

Precision immunotherapy for cholangiocarcinoma: Pioneering the use of human-derived anti-cMET single chain variable fragment in anti-cMET chimeric antigen receptor (CAR) NK cells.

Cholangiocarcinoma (CCA) presents a significant clinical challenge which is often identified in advanced stages, therby restricting the effectiveness of surgical interventions for most patients. The high incidence of cancer recurrence and resistance to chemotherapy further contribute to a bleak prognosis and low survival rates. To address this pressing need for effective therapeutic strategies, our study focuses on the development of an innovative cellular immunotherapy, specifically utilizing chimeric antigen receptor (CAR)-engineered natural killer (NK) cells designed to target the cMET receptor tyrosine kinase. In this investigation, we initiated the screening of a phage library displaying human single-chain variable fragment (ScFv) to identify novel ScFv molecules with specificity for cMET. Remarkably, ScFv11, ScFv72, and ScFv114 demonstrated exceptional binding affinity, confirmed by molecular docking analysis. These selected ScFvs, in addition to the well-established anti-cMET ScFvA, were integrated into a CAR cassette harboring CD28 transmembrane region-41BB-CD3ζ domains. The resulting anti-cMET CAR constructs were transduced into NK-92 cells, generating potent anti-cMET CAR-NK-92 cells. To assess the specificity and efficacy of these engineered cells, we employed KKU213A cells with high cMET expression and KKU055 cells with low cMET levels. Notably, co-culture of anti-cMET CAR-NK-92 cells with KKU213A cells resulted in significantly increased cell death, whereas no such effect was observed with KKU055 cells. In summary, our study identified cMET as a promising therapeutic target for CCA. The NK-92 cells, armed with the anti-cMET CAR molecule, have shown strong ability to kill cancer cells specifically, indicating their potential as a promising treatment for CCA in the future.

Dual action effects of ethyl-p-methoxycinnamate against dengue virus infection and inflammation via NF-κB pathway suppression.

Dengue virus (DENV) infection can lead to severe outcomes through a virus-induced cytokine storm, resulting in vascular leakage and inflammation. An effective treatment strategy should target both virus replication and cytokine storm. This study identified Kaempferia galanga L. (KG) extract as exhibiting anti-DENV activity. The major bioactive compound, ethyl-p-methoxycinnamate (EPMC), significantly reduced DENV-2 infection, virion production, and viral protein synthesis in HepG2 and A549 cells, with half-maximal effective concentration (EC50) values of 22.58 µM and 6.17 µM, and impressive selectivity indexes (SIs) of 32.40 and 173.44, respectively. EPMC demonstrated efficacy against all four DENV serotypes, targeting the replication phase of the virus life cycle. Importantly, EPMC reduced DENV-2-induced cytokines (IL-6 and TNF-α) and chemokines (RANTES and IP-10), as confirmed by immunofluorescence and immunoblot analyses, indicating inhibition of NF-κB activation. EPMC's role in preventing excessive inflammatory responses suggests it as a potential candidate for dengue treatment. Absorption, distribution, metabolism, excretion, and toxicity (ADMET) and drug-likeness for EPMC were predicted using SwissADME and ProTox II servers, showing good drug-like properties without toxicity. These findings highlight KG extract and EPMC as promising candidates for future anti-dengue therapeutics, offering a dual-action approach by inhibiting virus replication and mitigating inflammatory reactions.

Enhancing cholangiocarcinoma immunotherapy with adoptive T cells targeting HLA-restricted neoantigen peptides derived from driver gene mutations.

Precision immunotherapy, driven by genomic and bioinformatic advancements, has emerged as a promising and viable approach to combat cancer. Targeting neoantigens offers the advantage of specific immune responses with minimal off-tumor toxicity. In this study, we investigated the potential of adoptive T cells activated by HLA-restricted neoantigen peptides from driver gene mutations for treating cholangiocarcinoma (CCA), a highly aggressive cancer with poor prognosis and high mortality rates. Through whole exome sequencing of CCA cell lines, KKU-213A and KKU-100, we identified mutations in common driver genes and predicted corresponding HLA-restricted peptides. Peptides from KRAS, RNF43, and TP53 mutations exhibited strong binding affinity to HLA-A11, as validated through molecular docking and T2-cell binding assays. Dendritic cells (DCs) from healthy donors expressing HLA-A* 11:01, pulsed with individual or pooled peptides, showed comparable levels of costimulatory molecules (CD11c, CD40, CD86, and HLA-DR) to conventional DCs but higher expression of maturation markers, CD80 and CD86. Autologous HLA-A* 11:01-restricted T cells, activated by peptide-pulsed DCs, effectively lysed KKU-213A (HLA-A*11:01) cells, outperforming conventional tumor lysate-pulsed DCs. This effect was specific to HLA-A* 11:01-restricted T cells and not observed in KKU-100 (HLA-A*33:03) cells. Moreover, HLA-A* 11:01-restricted T cells exhibited elevated levels of IFN-gamma, granulysin, and granzyme B, indicating their potent anti-tumor capabilities. These findings underscore the specificity and efficiency of HLA-A* 11:01-restricted T cells targeting KRAS, RNF43, TP53 mutated CCA cells, and offer valuable insights for developing immunotherapeutic strategies and therapeutic peptide-vaccines for CCA treatment.

Anti-cancer effect of a phytochemical compound - 7R-acetylmelodorinol - against triple-negative breast cancer cells.

Triple-negative breast cancer (TNBC) is a highly aggressive subtype currently lacking effective treatment options. Consequently, novel and effective drugs or compounds are urgently needed to treat TNBC. Therefore, this study aimed to evaluate the potential of 7R-acetylmelodorinol (7R-AMDL), a phytochemical compound isolated from Xylopia pierrei Hance, a plant found in Thailand, as a novel therapeutic agent for TNBC. MTT and clonogenic assays showed that 7R-AMDL significantly reduced the survival of breast cancer cell lines, with a markedly potent effect on MDA-MB-231 cells. Flow cytometry showed that treating MDA-MB-231 cells with 7R-AMDL at the concentration of dose 8 µM significantly increased early and late apoptosis after 24 and 48 h compared to the control group (p < 0.0001). The highest tested 7R-AMDL dose upregulated the death receptors and their ligands, with extrinsic and intrinsic apoptosis pathways significantly activated via the caspase cascade, compared to the untreated group (p < 0.05). In addition, immunoblots showed decreased BCL2-like 1 (BCL2L1/Bcl-xL) expression (p < 0.0001). Furthermore, wound healing and Transwell assays showed that at a non-cytotoxic dose (≤4 µM), 7R-AMDL significantly inhibited the MDA-MB-231 cell migration and invasion. This reduction in cell migration was associated with decreased matrix metallopeptidase 9 (MMP-9) expression (p < 0.01) and nuclear factor kappa B (NF-κB) activation (p < 0.05). Altogether, 7R-AMDL has anti-cancer effects against TNBC and the potential to be further developed and evaluated for treating this disease.

Microalga Chlorella sp. extract induced apoptotic cell death of cholangiocarcinoma via AKT/mTOR signaling pathway.

Cancer is the leading cause of death worldwide. Drug resistance and relapse after current standard treatments frequently occur; thus, alternative and effective treatments are required. Algae and cyanobacteria are abundant organisms that serve as bioresources of nutrients/metabolites, which are attractive sources of numerous bioactive compounds for drug discovery. In the present study, we, therefore, investigated anti-cancer activities of crude polysaccharide and ethanolic extracts from Chlorella sp., Sargassum spp., and Spirulina sp. against cell lines of five top-leading cancers including lung cancer (A549), cervical cancer (Hela), breast cancer (MCF7), hepatocellular carcinoma (Huh7), and cholangiocarcinoma (CCA; KKU213A). Only ethanolic extracts of Chlorella sp. showed consistent inhibition of growth of all cancer cell types. CCA was the most sensitive to Chlorella sp. ethanolic extract with CC50 of 277.4, 400.5, and 313.4 µg/mL for KKU055, KKU100, and KKU213A cells, respectively. Flow cytometric analysis demonstrated that CCA cell death was triggered via apoptosis pathway in accompany with lowering procaspase-3, -8, and -9 and increasing caspase enzymatic activity in addition to reducing anti-apoptosis Bcl-2 protein. Interestingly, the treatment of the extract at 400 µg/mL greatly inhibited the AKT/mTOR survival signaling as evidenced by significant reduction of phosphorylated-AKT and phosphorylated-mTOR proteins. The presence of reported bioactive compounds, gallic acid, and lutein, were confirmed in Chlorella sp. extract by high-performance liquid chromatography. Gallic acid and lutein treatment caused a significant reduction of KKU055, KKU100, and KKU213A cell viability. This study demonstrated the anti-cancer effect of Chlorella sp. ethanolic extract to promote cancer cell death via inhibition of AKT/mTOR pathway.

Intraspecific Diversity and Pathogenicity of Bacillus thuringiensis Isolates from an Emetic Illness.

This study describes an emetic food-borne intoxication associated with a Bacillus cereus group species and the characterization of the bacterial isolates from the incident in aspects of molecular tying, genetic factors, cytotoxicity, and pathogenic mechanisms relating to emetic illness. Through the polyphasic identification approach, all seven isolates obtained from food and clinical samples were identified as Bacillus thuringiensis. According to multilocus sequence typing (MLST) analysis, intraspecific diversity was found within the B. thuringiensis isolates. Four allelic profiles were found, including two previously known STs (ST8 and ST15) and two new STs (ST2804 and ST2805). All isolates harbored gene fragments located in the cereulide synthetase (ces) gene cluster. The heat-treated culture supernatants of three emetic B. thuringiensis isolates, FC2, FC7, and FC8, caused vacuolation and exhibited toxicity to Caco-2 cells, with CC50 values of 56.57, 72.17, and 79.94 µg/mL, respectively. The flow cytometry with the Annexin V/PI assay revealed both apoptosis and necrosis mechanisms, but necrosis was the prominent mechanism that caused Caco-2 cell destruction by FC2, the most toxic isolate.

Effect of Holoptelea integrifolia (Roxb.) Planch. n-Hexane Extract and Its Bioactive Compounds on Wound Healing and Anti-Inflammatory Activity.

The stem bark of Holoptelea integrifolia (Roxb.) Planch. has been applied for the treatment of human cutaneous diseases as well as canine demodicosis in several countries. However, no detailed mechanistic studies have been reported to support their use. In this study, thin-layer chromatography and gas chromatography were used to screen phytochemicals from the fresh stem bark extract of H. integrifolia. We found the two major bioactive compounds, friedelin and lupeol, and their activity on wound healing was further investigated in keratinocytes. Both bioactive compounds significantly reduced wound area and increased keratinocyte migration by increasing matrix metalloproteinases-9 production. Subsequently, we found that the mRNA gene expressions of cadherin 1 and desmoglobin 1 significantly decreased, whereas the gene expression involved in keratinocyte proliferation and homeostasis (keratin-17) increased in compound-treated human immortalized keratinocytes cells. The expression of inflammatory genes (cyclooxygenase-2 and inducible nitric oxide synthase) and pro-inflammatory cytokine genes (tumor necrosis factor-alpha and interleukin-6) was reduced by treatment with n-hexane extract of H. integrifolia and its bioactive compounds. Our results revealed that H. integrifolia extract and its bioactive compounds, friedelin and lupeol, exhibit wound-healing activity with anti-inflammatory properties, mediated by regulating the gene expression involved in skin re-epithelialization.

Anti-CD19 chimeric antigen receptor T cells secreting anti-PD-L1 single-chain variable fragment attenuate PD-L1 mediated T cell inhibition.

Adoptive T cell therapy using second-generation anti-CD19 chimeric antigen receptor T cells (anti-CD19-CAR2-T) induced complete remission in many heavily pretreated patients with B cell acute lymphoblastic leukemia (B-ALL) or diffuse large B cell lymphoma (DLBCL). However, poor clinical efficacy was observed in treating aggressive B cell lymphomas (BCL). The limited T cell function was reported by programmed cell death protein 1 ligand (PD-L1) expressed on BCL cells bound to the PD-1 receptor on T cells. To overcome this problem, we generated anti-CD19-CAR4-T cells secreting anti-PD-L1 single-chain variable fragment (scFv), namely anti-CD19-CAR5-T cells, and evaluated their functions in vitro. Both anti-CD19-CAR-T cells contain an anti-CD19 scFv derived from a monoclonal antibody, FMC63, linked to CD28/4-1BB/CD27/CD3ζ. The secreting anti-PD-L1 scFv is derived from atezolizumab. Our results showed that secreted anti-PD-L1 scFv could bind to PD-L1 and block the binding of anti-PD-L1 monoclonal antibodies on PD-L1high tumor cells. Anti-CD19-CAR4-T and anti-CD19-CAR5-T cells efficiently killed CD19+ target tumor cells in two-dimensional (2D) and three-dimensional (3D) co-culture systems. However, anti-CD19-CAR5-T cells demonstrated superior proliferative ability. Interestingly, at a low effector (E) to target (T) ratio of 0.5:1, anti-CD19-CAR5-T cells showed higher cytotoxicity against CD19+/PD-L1high cells compared to that of anti-CD19-CAR4-T cells. The cytotoxicity of anti-CD19-CAR4-T cells against CD19+/PD-L1high could be restored by adding anti-PD-L1 scFv. Our findings demonstrate the combination antitumor efficiency of anti-CD19-CAR4-T cells and anti-PD-L1 scFv against CD19+/PD-L1high tumors. As such, anti-CD19-CAR5-T cells should be further investigated in vivo antitumor efficiency and clinical trials as a treatment for aggressive B cell lymphoma.

Genistein Sensitizes Human Cholangiocarcinoma Cell Lines to Be Susceptible to Natural Killer Cells.

Cholangiocarcinoma (CCA) is a lethal bile duct cancer, which has poor treatment outcomes due to its high resistance to chemotherapy and cancer recurrence. Activation of aberrant anti-apoptotic signaling pathway has been reported to be a mechanism of chemoresistance and immune escape of CCA. Therefore, reversal of anti-apoptotic signaling pathway represents a feasible approach to potentiate effective treatments, especially for CCA with high chemoresistance. In this study, we demonstrated the effects of genistein on reactivation of apoptosis cascade and increase the susceptibility of CCA cells to natural killer (NK-92) cells. Genistein at 50 and 100 µM significantly activated extrinsic apoptotic pathway in CCA cells (KKU055, KKU100, and KKU213A), which was evident by reduction of procaspase-8 and -3 expression. Pretreatment of CCA cells with genistein at 50 µM, but not NK-92 cells, significantly increased NK-92 cell killing ability over the untreated control, suggesting the ability of genistein to sensitize CCA cells. Interestingly, genistein treatment could greatly lower the expression of cFLIP, an anti-apoptotic protein involved in the immune escape pathway, in addition to upregulation of death receptors, Fas- and TRAIL-receptors, in CCA cells, which might be the underlying molecular mechanism of genistein to sensitize CCA to be susceptible to NK-92 cells. Taken together, this finding revealed the benefit of genistein as a sensitizer to enhance the efficiency of NK cell immunotherapy for CCA.

Combination gemcitabine and PD-L1xCD3 bispecific T cell engager (BiTE) enhances T lymphocyte cytotoxicity against cholangiocarcinoma cells.

Cholangiocarcinoma (CCA) is a lethal cancer with rapid progression and poor survival. Novel and more effective therapies than those currently available are, therefore, urgently needed. Our research group previously reported the combination of gemcitabine and cytotoxic T lymphocytes to be more effective than single-agent treatment for the elimination of CCA cells. However, gemcitabine treatment of CCA cells upregulates the expression of an immune checkpoint protein (programmed death-ligand 1 [PD-L1]) that consequently inhibits the cytotoxicity of T lymphocytes. To overcome this challenge and take advantage of PD-L1 upregulation upon gemcitabine treatment, we generated recombinant PD-L1xCD3 bispecific T cell engagers (BiTEs) to simultaneously block PD-1/PD-L1 signaling and recruit T lymphocytes to eliminate CCA cells. Two recombinant PD-L1xCD3 BiTEs (mBiTE and sBiTE contain anti-PD-L1 scFv region from atezolizumab and from a published sequence, respectively) were able to specifically bind to both CD3 on T lymphocytes, and to PD-L1 overexpressed after gemcitabine treatment on CCA (KKU213A, KKU055, and KKU100) cells. mBiTE and sBiTE significantly enhanced T lymphocyte cytotoxicity against CCA cells, especially after gemcitabine treatment, and their magnitudes of cytotoxicity were positively associated with the levels of PD-L1 expression. Our findings suggest combination gemcitabine and PD-L1xCD3 BiTE as a potential alternative therapy for CCA.

Cytotoxic T Cells Activated by Self-differentiated Monocyte-derived Dendritic Cells Against Multiple Myeloma Cells.

BACKGROUND/AIM: B cell maturation antigen (BCMA) is an ideal target for adoptive T cell therapy of multiple myeloma (MM). In this study, we evaluated self-differentiated monocyte-derived dendritic cells expressing BCMA (SD-DC-BCMA) to activate T cells for killing MM cells. MATERIALS AND METHODS: Lentivirus-modified SD-DC-BCMA harboring tri-cistronic cDNAs encoding granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-4 (IL-4), and BCMA was generated. Cytotoxicity of T cells activated by SD-DC-BCMA against MM cells was evaluated. RESULTS: T cells activated by SD-DC-BCMA exhibited a dose-dependent cytotoxicity against BCMA-expressing MM cells and produced high IFN-γ levels, compared to inactivated T cells or control T cells. A significantly higher killing ability of T cells activated by SD-DC-BCMA was further demonstrated in BCMA-overexpressing cells when compared with BCMA-negative cells. CONCLUSION: The potency of SD-DC-BCMA to activate T cells for antigen-specific cancer killing provides a framework for therapeutic application of adoptive T cell therapy in MM.

Doxorubicin sensitizes breast cancer cells to natural killer cells in connection with increased Fas receptors.

Breast cancer (BC) is the most common cancer in women. Although standard treatments are successful in patients with BC diagnosed at an early stage, an alternative treatment is required for patients with advanced­stage disease who do not respond to these treatments. The concept of using chemotherapy to sensitize cancer cells to become susceptible to immunotherapy was recently introduced and may be used as an alternative treatment for BC. The chemotherapeutic drug doxorubicin has been reported to sensitize cancer cells; however, the efficacy to sensitize the solid spheroids, in addition to its underlying mechanism regarding how doxorubicin sensitizes BC, has not previously been explored. In the present study, the effectiveness of a combined treatment of doxorubicin and natural killer­92 (NK­92) cells against BC in either 2D or 3D spheroid models, and its association with Fas receptor (FasR) expression, was demonstrated. The BC (MCF7) cell line expressing a higher level of FasR was more sensitive to NK­92 cell killing than the MDA­MB­231 cell line, which expressed a lower level of FasR. A sublethal dose of doxorubicin caused a significant improvement in NK cytotoxicity. Concordantly, a significant reduction in cell viability was observed in the doxorubicin­treated MCF7 spheroids. Notably, flow cytometric analysis revealed significantly increased FasR expression in the MCF7 cells, suggesting the underlying sensitization mechanism of doxorubicin in BC was related to the FasR upregulation. The present findings supported the use of combined doxorubicin and NK immunotherapy in BC treatment.

Triphala in Traditional Ayurvedic Medicine Inhibits Dengue Virus Infection in Huh7 Hepatoma Cells.

Traditional Triphala (three fruits), consisting of Phyllanthus emblica, Terminalia chebula, and Terminalia bellirica, presents a broad range of biological activities. However, its ability to inhibit dengue virus (DENV) infection has not been reported yet. Herein, the authors investigated the efficiency of three different Triphala formulations and its individual extract constituents to inhibit DENV infection. Treatment with T. bellirica extract or Triphala formulated with a high ratio of T. bellirica extract showed remarkable efficiency in significantly lowering DENV infection in Vero cells. Their effects were further studied in Huh7 cells, to address its potential ability in human cells. Treatment with 100 µg/mL of T. bellirica extract or Triphala resulted in an approximate 3000-fold or 1000-fold lowering of virus production, respectively. Furthermore, the treatment diminished IL-6 and CXCL-10 expressions, which are the hallmark of the cytokine storm phenomenon in DENV infection. The HPLC profiling demonstrated gallic acid as a major compound, the treatment by which showed its ability to effectively inhibit DENV infection after virus entry. Molecular docking demonstrated that gallic acid was able to interact with DENV NS5 protein, which could be one of Triphala's antiviral mechanism. This study offers Triphala formulation and its ingredient, T. bellirica extract, as a natural based pharmaceutical to be used in DENV infection treatment.