Water-soluble 4-(dimethylaminomethyl)heliomycin exerts greater antitumor effects than parental heliomycin by targeting the tNOX-SIRT1 axis and apoptosis in oral cancer cells.

The antibiotic heliomycin (resistomycin), which is generated from Streptomyces resistomycificus, has multiple activities, including anticancer effects. Heliomycin was first described in the 1960s, but its clinical applications have been hindered by extremely low solubility. A series of 4-aminomethyl derivatives of heliomycin were synthesized to increase water solubility; studies showed that they had anti-proliferative effects, but the drug targets remained unknown. In this study, we conducted cellular thermal shift assays (CETSA) and molecular docking simulations to identify and validate that heliomycin and its water-soluble derivative, 4-(dimethylaminomethyl)heliomycin (designated compound 4-dmH) engaged and targeted with sirtuin-1 (SIRT1) in p53-functional SAS and p53-mutated HSC-3 oral cancer cells. We further addressed the cellular outcome of SIRT1 inhibition by these compounds and found that, in addition to SIRT1, the water-soluble 4-dmH preferentially targeted a tumor-associated NADH oxidase (tNOX, ENOX2). The direct binding of 4-dmH to tNOX decreased the oxidation of NADH to NAD+ which diminished NAD+-dependent SIRT1 deacetylase activity, ultimately inducing apoptosis and significant cytotoxicity in both cell types, as opposed to the parental heliomycin-induced autophagy. We also observed that tNOX and SIRT1 were both upregulated in tumor tissues of oral cancer patients compared to adjacent normal tissues, suggesting their clinical relevance. Finally, the better therapeutic efficacy of 4-dmH was confirmed in tumor-bearing mice, which showed greater tNOX and SIRT1 downregulation and tumor volume reduction when treated with 4-dmH compared to heliomycin. Taken together, our in vitro and in vivo findings suggest that the multifaceted properties of water-soluble 4-dmH enable it to offer superior antitumor value compared to parental heliomycin, and indicated that it functions through targeting the tNOX-NAD+-SIRT1 axis to induce apoptosis in oral cancer cells.

The Use of Immune Regulation in Treating Head and Neck Squamous Cell Carcinoma (HNSCC).

Immunotherapy has emerged as a promising new treatment modality for head and neck cancer, offering the potential for targeted and effective cancer management. Squamous cell carcinomas pose significant challenges due to their aggressive nature and limited treatment options. Conventional therapies such as surgery, radiation, and chemotherapy often have limited success rates and can have significant side effects. Immunotherapy harnesses the power of the immune system to recognize and eliminate cancer cells, and thus represents a novel approach with the potential to improve patient outcomes. In the management of head and neck squamous cell carcinoma (HNSCC), important contributions are made by immunotherapies, including adaptive cell therapy (ACT) and immune checkpoint inhibitor therapy. In this review, we are focusing on the latter. Immune checkpoint inhibitors target proteins such as programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) to enhance the immune response against cancer cells. The CTLA-4 inhibitors, such as ipilimumab and tremelimumab, have been approved for early-stage clinical trials and have shown promising outcomes in terms of tumor regression and durable responses in patients with advanced HNSCC. Thus, immune checkpoint inhibitor therapy holds promise in overcoming the limitations of conventional therapies. However, further research is needed to optimize treatment regimens, identify predictive biomarkers, and overcome potential resistance mechanisms. With ongoing advancements in immunotherapy, the future holds great potential for transforming the landscape of oral tumor treatment and providing new hope for patients.

Luteolin Phosphate Derivatives Generated by Cultivating Bacillus subtilis var. Natto BCRC 80517 with Luteolin.

Luteolin (LUT), a plant-derived flavone, exhibits various bioactivities; however, the poor aqueous solubility hampers its applications. Here, we revealed bioconversion of LUT by Bacillus subtilis BCRC 80517, yielding three water-soluble phosphate conjugates. These derivatives were identified as luteolin 4'-O-phosphate (L4'P), luteolin 3'-O-phosphate (L3'P), and luteolin 7-O-phosphate (L7P) by LC-ESI-MS/MS and NMR. Besides, we found that Bacillus subtilis BCRC 80517 was able to convert different levels of LUT but showed a limited conversion rate. By observing bacterial morphology with transmission electron microscopy and confocal fluorescence microscopy, we found that LUT disrupted the bacterial membrane integrity, which explained the incomplete conversion. Additionally, we revealed a spontaneous intramolecular transesterification of L4'P to L3'P, the thermodynamically more stable form, under acidic conditions and proposed the possible mechanism involving a cyclic phosphate as the intermediate. This study provides insight into development of a potent structural modification strategy to enhance the solubility of LUT through biophosphorylation.

Celastrol, a plant-derived triterpene, induces cisplatin-resistance nasopharyngeal carcinoma cancer cell apoptosis though ERK1/2 and p38 MAPK signaling pathway.

BACKGROUND: Developing resistance to chemotherapeutic drugs has become a major problem in the management of nasopharyngeal carcinoma (NPC). To overcome this issue, use of natural plant products as chemosensitizers is gaining importance at a fast pace. HYPOTHESIS/PURPOSE: The present study was designed to evaluate the cytotoxic effect and mode of action of a natural pentacyclic triterpenoid, celastrol, on cisplatin-resistant NPC cells. RESULTS: Study results revealed that celastrol treatment significantly reduced the viability of NPC cells in dose and time dependent manners, as compared to untreated control cells. The cytotoxic effect of celastrol was mediated by cell cycle arrest at G2/M phase and induction of intrinsic and extrinsic apoptotic pathways. With further analysis, we observed that celastrol-induced activation of caspases was accompanied by increased phosphorylation of MAPK pathway proteins, p38, ERK1/2. CONCLUSION: Taken together, our observation provides a novel insight on use of a natural plant product, celastrol, in the management of chemoresistant NPC.

DNA Assembly in 3D Printed Fluidics.

The process of connecting genetic parts-DNA assembly-is a foundational technology for synthetic biology. Microfluidics present an attractive solution for minimizing use of costly reagents, enabling multiplexed reactions, and automating protocols by integrating multiple protocol steps. However, microfluidics fabrication and operation can be expensive and requires expertise, limiting access to the technology. With advances in commodity digital fabrication tools, it is now possible to directly print fluidic devices and supporting hardware. 3D printed micro- and millifluidic devices are inexpensive, easy to make and quick to produce. We demonstrate Golden Gate DNA assembly in 3D-printed fluidics with reaction volumes as small as 490 nL, channel widths as fine as 220 microns, and per unit part costs ranging from $0.61 to $5.71. A 3D-printed syringe pump with an accompanying programmable software interface was designed and fabricated to operate the devices. Quick turnaround and inexpensive materials allowed for rapid exploration of device parameters, demonstrating a manufacturing paradigm for designing and fabricating hardware for synthetic biology.

The development of a counterflow-assisted preconcentration technique in capillary electrophoresis electrospray-ionization mass spectrometry.

A preconcentration approach for CE-MS using counterflow-assisted electrokinetic injection was proposed. The proposed preconcentration method was based on a counterflow-compatible sheathless interface. The interface was fabricated using a capillary-assembled PDMS microdevice that allowed the application of a counterflow and provided liquid-film electrical conduction. During electrokinetic injection, a hydrodynamic counterflow was introduced into the separation capillary to retard the movement of the stacking boundary to the CE outlet. Accordingly, a long injection time was achieved without a loss of CE separation. With the use of reverse polarity mode and a dynamic polybrene-coated separation capillary (filled with a 1% formic acid solution), a counterflow-assisted electrokinetic injection of peptide samples (in a aqueous solution containing 1% ammonia and 50% methanol) for 10 min provided a sensitivity enhancement of 750-1480 and a detection limit of 20 pM for the five peptide standards. The proposed technique was applied to the analysis of low nanomolar concentrations myoglobin tryptic peptides.

Sheathless capillary electrophoresis electrospray ionization-mass spectrometry interface based on poly(dimethylsiloxane) membrane emitter and thin conducting liquid film.

This study develops a sheathless CE-MS interface using a robust PDMS membrane emitter and liquid-film electric conduction. A 3D mold was constructed for casting the device by using a one-step casting procedure. The interface consisted of a 125 µm-thick triangular emitter with a 50 µm-diameter microchannel, a conducting reservoir, and a 375 µm-diameter channel for assembling the separation capillary. The separation capillary was inserted into the 375 µm channel and connected to the emitter through the conducting reservoir. The electric contact for the CE outlet was established through a conductive liquid film in the space between the capillary terminus and the 375 µm channel. The one-step casting procedure and using a membrane emitter instead of a tapered emitter produced an easily fabricated and robust interface. A stable electrospray was obtained from 30 to 350 nL/min. Analyzing a five-peptide mixture in low-EOF (60 nL/min) and high-EOF (210 nL/min) conditions demonstrated the utility of the interface.

Capsaicin induces cell cycle arrest and apoptosis in human KB cancer cells.

BACKGROUND: Capsaicin, a pungent phytochemical in a variety of red peppers of the genus Capsicum, has shown an anti-proliferative effect on various human cancer cell lines. In contrast, capsaicin has also been considered to promote the growth of cancer cells. Thus, the effects of capsaicin on various cell types need to be explored. The anti-proliferative effects of capsaicin on human KB cancer cells are still unknown. Therefore, we examined the viability, cell cycle progression, and factors associated with apoptosis in KB cells treated with capsaicin. METHODS: The cell proliferation/viability and cytotoxicity of KB cells exposed to capsaicin were determined by a sulforhodamine B colorimetric assay and trypan blue exclusion. Apoptosis was detected by Hoechst staining and confirmed by western blot analysis of poly-(ADP-ribose) polymerase cleavage. Cell cycle distribution and changes of the mitochondrial membrane potential were analyzed by flow cytometry. Furthermore, the expression of caspase 3, 8 and 9 was evaluated by immunoblotting. RESULTS: We found that treatment of KB cells with capsaicin significantly reduced cell proliferation/viability and induced cell death in a dose-dependent manner compared with that in the untreated control. Cell cycle analysis indicated that exposure of KB cells to capsaicin resulted in cell cycle arrest at G2/M phase. Capsaicin-induced growth inhibition of KB cells appeared to be associated with induction of apoptosis. Moreover, capsaicin induced disruption of the mitochondrial membrane potential as well as activation of caspase 9, 3 and poly-(ADP-ribose) polymerase in KB cells. CONCLUSIONS: Our data demonstrate that capsaicin modulates cell cycle progression and induces apoptosis in human KB cancer cells through mitochondrial membrane permeabilization and caspase activation. These observations suggest an anti-cancer activity of capsaicin.

In vitro and in vivo anticancer effects of destruxin B on human colorectal cancer.

AIM: The study of the anticancer effects of destruxin B (DB) is rare and its anticancer mechanism remains unknown. The aim of this study was to test the in vitro and in vivo anticancer effects of DB, on human HT-29 colorectal cancer (CRC). MATERIALS AND METHODS: DB was isolated and characterized by high pressure liquid chromatography, electrospray ionization mass spectrometry and (1)H-nuclear magnetic resonance spectroscopy. (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to assess the effects of DB on HT-29 cells in vitro. The anticancer effects of DB were investigated in a murine xenograft model of human colon cancer. RESULTS: A significant inhibition of cell viability was observed with DB treatment in time- and dose-dependent manners. DB administered subcutaneously daily at 0.6-15 mg/kg was proven to be safe and effective in inhibiting the growth of CRC cells. Expression of Bax, cleaved poly (ADP-ribose) polymerase and active caspase-3 were observed with DB treatment and the increase in tumor volumes of treated groups were significantly (p<0.05) lower than those of the mock-treated group. CONCLUSION: DB has potential as a new therapeutic agent against human CRC.

Estimation of the measurement uncertainty in quantitative determination of ketamine and norketamine in urine using a one-point calibration method.

An approach was proposed for the estimation of measurement uncertainty for analytical methods based on one-point calibration. The proposed approach is similar to the popular multiple-point calibration approach. However, the standard deviation of calibration was estimated externally. The approach was applied to the estimation of measurement uncertainty for the quantitative determination of ketamine (K) and norketamine (NK) at a 100 ng/mL threshold concentration in urine. In addition to uncertainty due to calibration, sample analysis was the other major source of uncertainty. To include the variation due to matrix effect and temporal effect in sample analysis, different blank urines were spiked with K and NK and analyzed at equal time intervals within and between batches. The expanded uncertainties (k = 2) were estimated to be 10 and 8 ng/mL for K and NK, respectively.

Chiral electrokinetic chromatography-electrospray ionization-mass spectrometry using a double junction interface.

A double junction interface was utilized to preserve separation efficiency and alleviate ion suppression from sulfated ß-cyclodextrin (S-ß-CD) in electrokinetic chromatography-electrospray ionization-mass spectrometry. The utility of the approach was demonstrated by chiral EKC-MS analysis of dihydroxyphenylalanine and methyldihydroxyphenylalanine enantiomers using either low concentration (counter-migration mode; 0.1% S-ß-CD) or high concentration (carrier mode; 2% S-ß-CD). In the counter-migration mode, S-ß-CD anions were supplied continuously from the junction reservoir to the separation column so that the effective separation length was preserved. This interface is especially useful under carrier mode in which high concentration of S-ß-CD will migrate toward the ESI source. With the use of the double junction interface, the S-ß-CD exited the separation column will remain in the junction reservoir, whereas the analyte will flow toward the ESI source through a connecting column. As a result, no ion suppression was observed and the sensitivity was improved significantly.

Fast CEC-MS using poly(dimethylsiloxane) microinjector, short packed column, and low-sheath-flow interface.

A fast CEC-MS approach based on a microinjector and a short CEC column was developed. Poly(dimethylsiloxane) was used as the substrate for microinjector fabrication. A short capillary column (∼5 cm) packed with 5 µm octadecyl silica particles was inserted into the microinjector. The microinjector CEC device was interfaced to ESI-MS using a low-flow sheath liquid interface. The device delivers the advantages of sample introduction, pre-concentration, elution, and fast analysis as in chip-CEC yet avoids the difficulty of packing stationary material into the chip. The online pre-concentration and CEC-MS analysis capabilities of this device were demonstrated by analysis of a six-triazine mixture. A signal enhancement of 20-99-fold was achieved with a sample loading time of 180 s.

Staggered multistep elution solid-phase extraction capillary electrophoresis/tandem mass spectrometry: a high-throughput approach in protein analysis.

An approach based on staggered multistep elution solid-phase extraction (SPE) capillary electrophoresis/tandem mass spectrometry (CE/MS/MS) was developed in the analysis of digested protein mixtures. On-line coupling of SPE with CE/MS was achieved using a two-leveled two-cross polydimethylsiloxane (PDMS)-based interface. Multistep elution SPE was used prior to CE to provide an additional dimension of separation, thus extending the separation capacity for the peptide mixture analysis. By decreasing in the number of co-eluting peptides, problems stemming from ionization suppression and finite MS/MS duty cycle were reduced. As a result, sequence coverage increased significantly using multistep elution SPE-CE/MS/MS compared to one-step elution SPE-CE/MS/MS in the analysis of a single protein tryptic digest (49% vs. 18%) and a six protein tryptic digest (22-71% vs. 10-44%). A staggered CE method was incorporated to increase the throughput. The electropherograms of consecutive CE runs were partially overlapped by injecting the sample plug at a fixed time interval. With the use of a 5 min injection interval, slightly poor results were obtained in comparison with the sequential CE method while the total analysis time was reduced to 28%.

Development of a liquid-junction/low-flow interface for phosphate buffer capillary electrophoresis mass spectrometry.

To alleviate ion suppression from phosphate buffer and to preserve separation integrity, a new capillary electrophoresis mass spectrometry (CE-MS) interface was developed. The interface consisted of a low-flow interface and a liquid junction. In this design, both the inlet reservoir and the liquid-junction reservoir were filled with phosphate running buffer. Because the phosphate anions in the column migrated toward the inlet reservoir (away from the electrospray ionization (ESI) source) the problem of ion suppression in ESI was avoided. The liquid junction was incorporated to eliminate issues of degraded separation observed when sheath liquid interfaces use different buffers for separation and MS analysis attributed to differences in anion velocity. The utility of the interface was demonstrated by the analysis of antihistamines at pH 3.5 and the analysis of perfluorocarboxylic acid at pH 9.5.