Cordycepin Sensitizes Cholangiocarcinoma Cells to Be Killed by Natural Killer-92 (NK-92) Cells.

Immunotherapy harnessing immune functions is a promising strategy for cancer treatment. Tumor sensitization is one approach to enhance tumor cell susceptibility to immune cell cytotoxicity that can be used in combination with immunotherapy to achieve therapeutic efficiency. Cordycepin, a bioactive compound that can be extracted from some Cordyceps spp. has been reported to effectively inhibit tumor growth, however, the mechanism of its tumor sensitization activity that enhances immune cell cytotoxicity is unknown. In the present study, we investigated the potency of cordycepin to sensitize a lethal cancer, cholangiocarcinoma (CCA), to natural killer (NK) cells. Treatment with cordycepin prior to and during co-culturing with NK-92 cells significantly increased cell death of KKU-213A as compared to solitary cordycepin or NK treatment. Moreover, sensitization activity was also observed in the combination of NK-92 cells and Cordyceps militaris extract that contained cordycepin as a major component. The cordycepin treatment remarkably caused an increase in TRAIL receptor (DR4 and DR5) expression in KKU-213A, suggesting the possible involvement of TRAIL signaling in KKU-213A sensitization to NK-92 cells. In conclusion, this is the first report on the sensitization activity of cordycepin on CCA cells to NK cytotoxicity, which supports that cordycepin can be further developed as an alternate immunomodulating agent.

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.

Generation of two patient specific GABRD variants and their isogenic controls for modeling epilepsy.

Developmental and epileptic encephalopathies (DEEs) are early-onset conditions that cause intractable seizures and developmental delays. Missense variants in Gamma-aminobutyric acid type A receptor (GABAAR) subunits commonly cause DEEs. Ahring et al. (2022) showed a variant in the gene that encodes the delta subunit (GABRD) is strongly associated with the gain-of-function of extrasynaptic GABAAR. Here, we report the generation of two patient-specific human induced pluripotent stem cells (hiPSC) lines with (i) a de novo variant and (ii) a maternal variant, both for the pathogenic GABRD c.872 C>T, (p.T291I). The variants in the generated cell line were corrected using the CRISPR-Cas9 gene editing technique (respective isogenic control lines).

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.

Generation of eight hiPSCs lines from two pathogenic variants in CACNA1A using the CRISPR-Cas9 gene editing technology.

Developmental and epileptic encephalopathies (DEEs) are rare severe neurodevelopmental disorders with a cumulative incidence of 1:6.000 live births. Many epileptic conditions arise from single nucleotide variants in CACNA1A (calcium voltage-gated channel subunit alpha1 A), encoding the CaV2.1 calcium channel subunit. Human induced pluripotent stem cells (hiPSCs) are an optimal choice for modeling DEEs, as they can be differentiated in vitro into diverse neuronal subpopulations. Here, we report the generation of hiPSC lines with two pathogenic CACNA1A variants c.1767C > T, p. (Arg589Cys), referred to as R589C and c. 2139G > A, p.(Ala713Thr), referred to as A713T, previously associated with epilepsy. The variants were introduced into a hiPSC line from a healthy individual via CRISPR-Cas9 gene editing technology.

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.

A comprehensive ethnic-based analysis of alpha thalassaemia allelle frequency in northern Thailand.

Alpha (α)-thalassaemia is one of the most prevalent hereditary blood disorders, commonly affecting Southeast Asian people, with the highest incidence (30-40%) being seen in northern Thailand. However, this high incidence was estimated without consideration of the variations between ethnic populations and the geographical location of the populations. To address this issue, a total of 688 samples from 13 different northern Thai ethnic groups (30 villages) categorized into three linguistic groups were genotyped for deletional alpha-thalassaemia (-α3.7, -α4.2, --SEA and --THAI) and/or non-deletional alpha-thalassaemia (αCS and αPS) via multiplex gap-PCR and dot-blot hybridization, respectively. Alpha+(-α3.7, -α4.2, αCS and αPS) and alpha°-thalassaemia (--SEA and --THAI) allele frequencies (with 95% Confidence Interval) were the highest in the Sino-Tibetan group [0.13 (0.08-0.18)] and the Tai-Kadai group [0.03 (0.02-0.05)], respectively. With regards to ethnicity, the varying allele frequency of α+ and α°-thalassaemia amongst a variety of ethnic groups was observed. The highest α+-thalassaemia allele frequency was found in the Paluang [0.21 (0.10-0.37)] while α°-thalassaemia allele frequency was the highest in the Yuan [0.04 (0.01-0.10)]. These detailed results of alpha thalassaemia allele frequency and genetic diversity amongst the northern Thai ethnic groups demonstrate the need for ethnicity based thalassaemia prevention programs.