Pickering Emulsion of Oleoresin from Dipterocarpus alatus Roxb. ex G. Don and Its Antiproliferation in Colon (HCT116) and Liver (HepG2) Cancer Cells.

Oleoresin of Dipterocarpus alatus Roxb. ex G. Don (DA) has been traditionally used for local medicinal applications. Several in vitro studies have indicated its pharmacological potential. However, the low water solubility hinders its use and development for pharmaceutical purposes. The study aimed to (1) formulate oil-in-water (o/w) Pickering emulsions of DA oleoresin and (2) demonstrate its activities in cancer cells. The Pickering emulsions were formulated using biocompatible carboxylated cellulose nanocrystal (cCNC) as an emulsifier. The optimized emulsion comprised 3% (F1) and 4% (v/v) (F2) of oleoresin in 1% cCNC and 0.1 M NaCl, which possessed homogeneity and physical stability compared with other formulations with uniform droplet size and low viscosity. The constituent analysis indicated the presence of the biomarker dipterocarpol in both F1 and F2. The pharmacological effects of the two emulsions were demonstrated in vitro against two cancer cell lines, HepG2 and HCT116. Both F1 and F2 suppressed cancer cell viability. The treated cells underwent apoptosis, as demonstrated by distinct nuclear morphological changes in DAPI-stained cells and Annexin V/PI-stained cells detected by flow cytometry. Our study highlights the prospect of Pickering emulsions for oleoresin, emphasizing enhanced stability and potential pharmacological advantages.

Probing the stability and quality of the cellulose-based Pickering emulsion containing sesamolin-enriched sesame oil by chemometrics-assisted ATR-FTIR spectroscopy.

This study presents the employment of Fourier transform infrared (FTIR) spectroscopy with attenuated total reflection and principal component analysis (PCA) to analyze the stability of a Pickering emulsion stabilized by carboxylated-cellulose nanocrystal (cCNC) comprising sesame oil phases with or without sesamolin. FTIR measurements identified an intermolecular hydrogen bond between the ester group of the triglyceride and the carboxyl group of the cCNC to create the emulsion droplet. The spectral bands from the hydroxyl group vibration (3700-3050 cm-1), carbonyl (1744 cm-1), CO groups of the ester triglyceride and cCNC (1160-998 cm-1) markedly discriminated between stabilized and destabilized emulsions. The PCA of FTIR spectra detected the change of molecular interaction during storage according to creaming, aggregation, and coalescence and changes in physicochemical parameters such as droplet size, refractive index, and zeta potential. Hence, PCA enabled the observation of the destabilization of emulsion in real-time.

Macromolecules with predominant ß-pleated sheet proteins in extracellular vesicles released from Raphanus sativus L. var. caudatus Alef microgreens induce DNA damage-mediated apoptosis in HCT116 colon cancer cells.

Plant-derived bioactive macromolecules (i.e., proteins, lipids, and nucleic acids) were prepared as extracellular vesicles (EVs). Plant-derived EVs are gaining pharmaceutical research interest because of their bioactive components and delivery properties. The spherical nanosized EVs derived from Raphanus sativus L. var. caudatus Alef microgreens previously showed antiproliferative activity in HCT116 colon cancer cells from macromolecular compositions (predominantly proteins). To understand the mechanism of action, the biological activity studies, i.e., antiproliferation, cellular biochemical changes, DNA conformational changes, DNA damage, apoptotic nuclear morphological changes, apoptosis induction, and apoptotic pathways, were determined by neutral red uptake assay, synchrotron radiation-based Fourier transform infrared microspectroscopy, circular dichroism spectroscopy, comet assay, 4',6-diamidino-2-phenylindole (DAPI) staining, flow cytometry, and caspase activity assay, respectively. EVs inhibited HCT116 cell growth in concentration- and time-dependent manners, with a half-maximal inhibitory concentration of 675.4 ± 33.8 µg/ml at 48 h and a selectivity index of 1.5 ± 0.076. HCT116 treated with EVs mainly changed the cellular biochemical compositions in the nucleic acids and carbohydrates region. The DNA damage caused no changes in DNA conformation. The apoptotic nuclear morphological changes were associated with the increased apoptotic cell population. The apoptotic cell death was induced by both extrinsic and intrinsic pathways. EVs have potential as antiproliferative bioparticles.

Stacked ensemble learning on HaCaT cytotoxicity for skin irritation prediction: A case study on dipterocarpol.

Skin irritation is an adverse effect associated with various substances, including chemicals, drugs, or natural products. Dipterocarpol, extracted from Dipterocarpus alatus, contains several skin benefits notably anticancer, wound healing, and antibacterial properties. However, the skin irritation of dipterocarpol remains unassessed. Quantitative structure-activity relationship (QSAR) is a recommended tool for toxicity assessment involving less time, money, and animal testing to access unavailable acute toxicity data. Therefore, our study aimed to develop a highly accurate machine learning-based QSAR model for predicting skin irritation. We utilized a stacked ensemble learning model with 1064 chemicals. We also adhered to the recommendations from the OECD for QSAR validation. Subsequently, we used the proposed model to explore the cytotoxicity of dipterocarpol on keratinocytes. Our findings indicate that the model displayed promising statistical quality in terms of accuracy, precision, and recall in both 10-fold cross-validation and test datasets. Moreover, the model predicted that dipterocarpol does not have skin irritation, which was confirmed by the cell-based assay. In conclusion, our proposed model can be applied for the risk assessment of skin irritation in untested compounds that fall within its applicability domain. The web application of this model is available at https://qsarlabs.com/#stackhacat.

StackBRAF: A Large-Scale Stacking Ensemble Learning for BRAF Affinity Prediction.

The B-rapidly accelerated fibrosarcoma (BRAF) is a proto-oncogene that plays a vital role in cell signaling and growth regulation. Identifying a potent BRAF inhibitor can enhance therapeutic success in high-stage cancers, particularly metastatic melanoma. In this study, we proposed a stacking ensemble learning framework for the accurate prediction of BRAF inhibitors. We obtained 3857 curated molecules with BRAF inhibitory activity expressed as a predicted half-maximal inhibitory concentration value (pIC50) from the ChEMBL database. Twelve molecular fingerprints from PaDeL-Descriptor were calculated for model training. Three machine learning algorithms including extreme gradient boosting, support vector regression, and multilayer perceptron were utilized for constructing new predictive features (PFs). The meta-ensemble random forest regression, called StackBRAF, was created based on the 36 PFs. The StackBRAF model achieves lower mean absolute error (MAE) and higher coefficient of determination (R2 and Q2) than the individual baseline models. The stacking ensemble learning model provides good y-randomization results, indicating a strong correlation between molecular features and pIC50. An applicability domain of the model with an acceptable Tanimoto similarity score was also defined. Moreover, a large-scale high-throughput screening of 2123 FDA-approved drugs against the BRAF protein was successfully demonstrated using the StackBRAF algorithm. Thus, the StackBRAF model proved beneficial as a drug design algorithm for BRAF inhibitor drug discovery and drug development.

Prediction of KRASG12C inhibitors using conjoint fingerprint and machine learning-based QSAR models.

Kirsten rat sarcoma virus G12C (KRASG12C) is the major protein mutation associated with non-small cell lung cancer (NSCLC) severity. Inhibiting KRASG12C is therefore one of the key therapeutic strategies for NSCLC patients. In this paper, a cost-effective data driven drug design employing machine learning-based quantitative structure-activity relationship (QSAR) analysis was built for predicting ligand affinities against KRASG12C protein. A curated and non-redundant dataset of 1033 compounds with KRASG12C inhibitory activity (pIC50) was used to build and test the models. The PubChem fingerprint, Substructure fingerprint, Substructure fingerprint count, and the conjoint fingerprint-a combination of PubChem fingerprint and Substructure fingerprint count-were used to train the models. Using comprehensive validation methods and various machine learning algorithms, the results clearly showed that the XGBoost regression (XGBoost) achieved the highest performance in term of goodness of fit, predictivity, generalizability and model robustness (R2 = 0.81, Q2CV = 0.60, Q2Ext = 0.62, R2 - Q2Ext = 0.19, R2Y-Random = 0.31 ± 0.03, Q2Y-Random = -0.09 ± 0.04). The top 13 molecular fingerprints that correlated with the predicted pIC50 values were SubFPC274 (aromatic atoms), SubFPC307 (number of chiral-centers), PubChemFP37 (≥1 Chlorine), SubFPC18 (Number of alkylarylethers), SubFPC1 (number of primary carbons), SubFPC300 (number of 1,3-tautomerizables), PubChemFP621 (N-C:C:C:N structure), PubChemFP23 (≥1 Fluorine), SubFPC2 (number of secondary carbons), SubFPC295 (number of C-ONS bonds), PubChemFP199 (≥4 6-membered rings), PubChemFP180 (≥1 nitrogen-containing 6-membered ring), and SubFPC180 (number of tertiary amine). These molecular fingerprints were virtualized and validated using molecular docking experiments. In conclusion, this conjoint fingerprint and XGBoost-QSAR model demonstrated to be useful as a high-throughput screening tool for KRASG12C inhibitor identification and drug design.

Bioactive Compounds, Antioxidant Activities, and HPLC Analysis of Nine Edible Sprouts in Cambodia.

The non-nutritional health benefits of sprouts are unconfirmed. Thus, nine sprout methanolic extracts were tested for phytoconstituents and antioxidant activity. The TPC, TCC, TFC, TAC, and TALC were measured. ABTS and DPPH radical scavenging and ferric-reducing antioxidant power assays were used to assess the antioxidant activity. HPLC detected gallic acid, vanillin, syringic acid, chlorogenic acid, caffeic acid, and rutin in the extracts. The sprout extracts contained six compounds, with caffeic acid being the most abundant. Gallic acid, syringic acid, chlorogenic acid, caffeic acid, vanillin, and rutin were highest in soybean, black sesame, mustard, sunflower, white radish, and black sesame sprouts, respectively. Sunflower sprouts had the highest level of TCC while soybean sprouts had the highest level of TFC, Taiwanese morning glory had the highest level of TPC, mustard sprouts had the highest level of TALC, and black sesame sprouts had the highest level of TAC. Taiwanese morning glories scavenged the most DPPH and ABTS radicals. Colored and white radish sprouts had similar ferric-reducing antioxidant power. Antioxidation mechanisms varied by compound. Our findings demonstrated that sprouts have biological effects, and their short time for mass production offers an alternative food source for health benefits, and that they are useful for future research development of natural products and dietary supplements.

Thai Rat-Tailed Radish Prevents Hepatocarcinogenesis in Rats by Blocking Mutagenicity, Inducing Hepatic Phase II Enzyme, and Decreasing Hepatic Pro-Inflammatory Cytokine Gene Expression.

Raphanus sativus L. var. caudatus Alef (RS) is an indigenous Thai plant with nutritional and medicinal values such as anticancer activity, but only in vitro. The chemopreventive effects of RS were, therefore, investigated in the initial stage of hepatocarcinogenesis in rats. Diethylnitrosamine (DEN), a carcinogen, was intraperitoneally injected into rats to induce liver cancer. Along with the DEN injection, either aqueous (RS-H2O) or dichloromethane (RS-DCM) extract was administered orally. Immunohistochemistry was used to detect glutathione S-transferase placental (GST-P) positive foci and apoptotic cells in rat livers as indicators of initial-stage carcinogenesis. The underlying mechanisms of chemoprevention were investigated with (a) antimutagenic activity, (b) hepatic phase II enzyme induction, and (c) hepatic pro-inflammatory cytokine gene expression. The results showed that RS-DCM was more potent than RS-H2O in decreasing GST-P positive foci and apoptotic cells induced by DEN. The mechanisms of RS-DCM (phenolics and sulforaphene contents) against liver carcinogenesis (1) block the activity of carcinogens; (2) elevate phase II detoxifying enzymes; and (3) suppress the pro-inflammatory gene expression. RS-H2O (phenolics contents), in contrast, only decreases pro-inflammatory gene expression. In conclusion, the RS extract consisting of phenolics and isothiocyanates exerted significant chemopreventive activity against DEN-induced liver carcinogenesis.

Controllability, antiproliferative activity, Ag+ release, and flow behavior of silver nanoparticles deposited onto cellulose nanocrystals.

Silver nanoparticles (AgNPs)/carboxylated cellulose nanocrystals (Ag-cCNC) from Eucalyptus pulp were prepared using a three-step process. The cCNC were synthesized by oxidation of CNC from Eucalyptus pulp with ammonium persulfate, followed by a hydrothermal reaction to form Ag-cCNC. The Ag-cCNC was then characterized with respect to Ag+ release, flow behavior, and anticancer activity for potential applications in biomedicine and drug delivery. AgNPs with particle sizes in the range of 16.25 ± 7.83 to 21.84 ± 7.21 nm were uniformly embedded on the surface of the cCNC. The Ag-cCNC exhibited a slow and controllable release of Ag+ at a rate of 0.02 % per day for 28 days. Ag+ release was best described by the Korsmeyer-Peppas model based on non-Fickian diffusion. The Ag-cCNC at 200 µg/mL exerted antiproliferative activity in MCF-7 human breast cancer cells with 1.01 % ± 0.35 % cell viability and was non-toxic against normal Vero cells with 90 % viability. In contrast, the chemotherapeutic drug melphalan exhibited cytotoxic effects against both MCF-7 and Vero cells. The Ag-cCNC samples showed shear thinning properties with a pseudoplastic fluid behavior, indicating that Ag-cCNCs are suitable for drug delivery by injection.

Extracellular vesicles derived from microgreens of Raphanus sativus L. var. caudatus Alef contain bioactive macromolecules and inhibit HCT116 cells proliferation.

Extracellular vesicles (EVs) are phospholipid bilayer vesicles released from cells, containing natural cargos. Microgreens of Raphanus sativus L. var. caudatus Alef were used in this study as the source of EVs. EVs were isolated by differential centrifugation. The physical properties were determined by dynamic light scattering (DLS) and electron microscopy. The biological and chemical composition were studied by Fourier-transform infrared (FTIR) microspectroscopy and high-performance liquid chromatography analysis, respectively. EVs had a median size of 227.17 and 234.90 ± 23.30 nm determined by electron microscopy and DLS, respectively with a polydispersity index of 0.293 ± 0.019. Electron microscopy indicated the intact morphology and confirmed the size. The FTIR spectra revealed that EVs are composed of proteins as the most abundant macromolecules. Using a curve-fitting analysis, ß-pleated sheets were the predominant secondary structure. Notably, the micromolecular biomarkers were not detected. EVs exerted anti-cancer activity on HCT116 colon cancer over Vero normal cells with an IC50 of 448.98 µg/ml and a selectivity index of > 2.23. To conclude, EVs could be successfully prepared with a simple and effective isolation method to contain nano-sized macromolecules possessing anti-cancer activity.

Development of Sesamol Carbamate-L-Phenylalanine Prodrug Targeting L-Type Amino Acid Transporter1 (LAT1) as a Potential Antiproliferative Agent against Melanoma.

Sesamol is a compound reported to have anti-melanogenesis and anti-melanoma actions. Sesamol, however, has low intracellular drug concentration and fast excretion, which can limit its benefits in the clinic. To overcome this drawback and increase intracellular delivery of sesamol into the target melanoma, research has focused on L-type amino acid transporter 1 (LAT1)-mediated prodrug delivery into melanoma cells. The sesamol prodrug was designed by conjugating sesamol with L-phenylalanine at the para position with a carbamate bond. LAT1 targeting was evaluated vis-à-vis a competitive [14C]-leucine uptake inhibition. The sesamol prodrug has a higher [14C]-leucine uptake inhibition than sesamol in human LAT1-transfected HEK293 cells. Moreover, the sesamol prodrug was taken up by LAT1-mediated transport into SK-MEL-2 cells more effectively than sesamol. The sesamol prodrug underwent complete hydrolysis, releasing the active sesamol at 72 h, which significantly exerted its cytotoxicity (IC50 of 29.3 µM) against SK-MEL-cells more than sesamol alone. Taken together, the strategy for LAT1-mediated prodrug delivery has utility for the selective uptake of sesamol, thereby increasing its intracellular concentration and antiproliferation activity, targeting melanoma SK-MEL-2 cells that overexpress the LAT1 protein. The sesamol prodrug thus warrants further evaluation in an in vivo model.

Dipterocarpol in Oleoresin of Dipterocarpus alatus Attributed to Cytotoxicity and Apoptosis-Inducing Effect.

Dipterocarpus alatus Roxb. ex G. Don is widely found in Southeast Asia. Its oleo-resin has reportedly been used in biodiesel production. Two different biodiesel production processes produce resinous byproducts, namely degumming (DG) and distillation (DT). Gas chromatography-mass spectrometry identified sesquiterpenes and triterpenes in oleo-resin, DG, and DT; and long-chain hydrocarbons in oleo-resin. High-performance liquid chromatography detected dipterocarpol as a marker compound, with the highest to lowest amounts detected in DG, DT, and oleo-resin, respectively. Oleo-resin, DG, and DT exerted more cytotoxicity than dipterocarpol, and melphalan, a chemotherapeutic drug. Oleo-resin, DG, and DT exerted cytotoxicity to a different degree in T cell leukemia (Jurkat), cervical adenocarcinoma (HeLa), and human hepatocellular carcinoma (HepG2) cells, while the highest selectivity was found in the Jurkat cells compared to the non-cancer Vero cells. Dipterocarpol exhibited the highest cytotoxicity in HepG2 cells and the lowest cytotoxicity in Jurkat cells. Oleo-resin, DG, and DT induced apoptosis in Jurkat cells. In oleo-resin, DG, and DT, dipterocarpol and other compounds may act in synergy leading to cytotoxicity and an apoptosis-inducing effect. Oleo-resin, DG, and DT could be potential sources for anticancer agents. Dipterocarpol could serve as a biomarker for follow ups on the anticancer activity of a sample from D. alatus.

Multiple Bioactivities of Manihot esculenta Leaves: UV Filter, Anti-Oxidation, Anti-Melanogenesis, Collagen Synthesis Enhancement, and Anti-Adipogenesis.

The cassava root is an important global agro-industrial crop that yields cassava leaf as a left-over co-product of interest for further development as a sustainable resource of health and cosmeceutical active compounds. This work aimed to investigate the cosmeceutical potential and chemical composition of an ethanolic cassava leaf extract (BM). rutin, apigenin, and kaempferol were found to be major constituents via HPLC-DAD UV analysis. Interestingly, the multiple beneficial bioactivities of BM for cosmeceutical applications were manifested in a dose-dependent manner, including anti-oxidation in a 2,2-diphenyl-1-picrylhydrazyl assay, anti-melanogenesis in B16 melanoma cells, collagen synthesis enhancement in human fibroblasts, and anti-adipogenesis in 3T3-L1 adipocytes. Furthermore, the potential of the collagen synthesis enhancement of BM and rutin was significant when compared to ascorbic acid. Additionally, a UV filter property comparable to BEMT with characteristics of board spectral absorption and constant high absorptivity throughout all UV wavelength ranges was exhibited by UV-visible spectrophotometric analysis. In conclusion, the cassava leaf was found to be a potential natural cosmeceutical active agent with multiple cosmeceutical-related bioactivities with respect to a substantial composition of bioactive flavonols. These obtained data will support and encourage the further study and development of cassava leaves as potential economic and sustainable sources of bioactive agents for health and cosmeceutical applications.

Route of intracellular uptake and cytotoxicity of sesamol, sesamin, and sesamolin in human melanoma SK-MEL-2 cells.

The intracellular uptake concentration determines drug absorption, drug activity, and toxicity. Sesamol, sesamin, and sesamolin are promising bioactive components from Sesame indicum L. Their respective intracellular uptake pathway and cytotoxicity were evaluated using melanoma and non-cancerous cells. Quantitative structure-activity relationship (QSAR) models were built to identify the molecular features affecting drug uptake in cells. The respective intracellular uptake pathway for sesamol vs. sesamin and sesamolin was carrier-mediated vs. passive transport. Topological polar surface area (PSA) and 2D autocorrections increase the intracellular concentration (C/M ratio) of these compounds. Sesamol has the lowest C/M ratio compared to sesamin and sesamolin, but only sesamol inhibits the cell viability of melanoma and provides an inhibition concentration at 50% (IC50) against melanoma cells. The slightly aqueous solubility of sesamin and sesamolin, therefore, limits testing of their cytotoxicity. In conclusion, sesamol has the potential to inhibit melanoma cell growth, but requires improvement of the C/M ratio to increase its physicochemical properties. Thus, in order to investigate the cytotoxicity of sesamin and sesamolin against melanoma cells a solubility enhancer is needed.

Drug Repurposing against KRAS Mutant G12C: A Machine Learning, Molecular Docking, and Molecular Dynamics Study.

The Kirsten rat sarcoma viral G12C (KRASG12C) protein is one of the most common mutations in non-small-cell lung cancer (NSCLC). KRASG12C inhibitors are promising for NSCLC treatment, but their weaker activity in resistant tumors is their drawback. This study aims to identify new KRASG12C inhibitors from among the FDA-approved covalent drugs by taking advantage of artificial intelligence. The machine learning models were constructed using an extreme gradient boosting (XGBoost) algorithm. The models can predict KRASG12C inhibitors well, with an accuracy score of validation = 0.85 and Q2Ext = 0.76. From 67 FDA-covalent drugs, afatinib, dacomitinib, acalabrutinib, neratinib, zanubrutinib, dutasteride, and finasteride were predicted to be active inhibitors. Afatinib obtained the highest predictive log-inhibitory concentration at 50% (pIC50) value against KRASG12C protein close to the KRASG12C inhibitors. Only afatinib, neratinib, and zanubrutinib covalently bond at the active site like the KRASG12C inhibitors in the KRASG12C protein (PDB ID: 6OIM). Moreover, afatinib, neratinib, and zanubrutinib exhibited a distance deviation between the KRASG2C protein-ligand complex similar to the KRASG12C inhibitors. Therefore, afatinib, neratinib, and zanubrutinib could be used as drug candidates against the KRASG12C protein. This finding unfolds the benefit of artificial intelligence in drug repurposing against KRASG12C protein.

An Insight into Sesamolin: Physicochemical Properties, Pharmacological Activities, and Future Research Prospects.

Sesame seeds are rich in lignan content and have been well-known for their health benefits. Unlike the other sesame lignan compounds (i.e., sesamin and sesamol), the study of the pharmacological activity of sesamolin has not been explored widely. This review, therefore, summarizes the information related to sesamolin's pharmacological activities, and the mechanism of action. Moreover, the influence of its physicochemical properties on pharmacological activity is also discussed. Sesamolin possessed neuroprotective activity against hypoxia-induced reactive oxygen species (ROS) and oxidative stress in neuron cells by reducing the ROS and inhibiting apoptosis. In skin cancer, sesamolin exhibited antimelanogenesis by affecting the expression of the melanogenic enzymes. The anticancer activity of sesamolin based on antiproliferation and inhibition of migration was demonstrated in human colon cancer cells. In addition, treatment with sesamolin could stimulate immune cells to enhance the cytolytic activity to kill Burkitt's lymphoma cells. However, the toxicity and safety of sesamolin have not been reported. And there is also less information on the experimental study in vivo. The limited aqueous solubility of sesamolin becomes the main problem, which affects its pharmacological activity in the in vitro experiment and clinical efficacy. Therefore, solubility enhancement is needed for further investigation and determination of its pharmacological activity profiles. Since there are fewer reports studying this issue, it could become a future prospective research opportunity.

Chemopreventive Effect of Cratoxylum formosum (Jack) ssp. pruniflorum on Initial Stage Hepatocarcinogenesis in Rats.

Cratoxylum formosum ssp. pruniflorum (Kurz) Gogelein (CP) is an indigenous plant found mainly in southeast Asia. Several in vitro studies have confirmed its activity against hepatocellular carcinoma; however, in vivo studies of the effect of CP on liver cancer are needed. This study investigated the effect of CP on early-stage hepatocarcinogenesis in rat liver when using diethylnitrosamine (DEN) as a carcinogen. Immunohistochemistry was used to detect (a) upregulation of glutathione S-transferase placental (GST-P) positive foci, (b) the proliferating cell nuclear antigen PCNA, and (c) apoptotic cells in the liver as indicators of early-stage carcinogenesis. Immunohistochemical parameters were observed in rats given CP orally following DEN injection. Rats given DEN presented overexpression of GST-P positive foci, PCNA, and apoptotic cells, indicating the formation of cancerous tissues, and these effects were diminished by CP treatment. CP thus inhibited hepatocarcinogenic effects in an animal model. These results could help plan further in vivo studies and support the use of CP to prevent processes that promote the pathogenesis of hepatocellular carcinoma in humans.

Pinus kesiya Royle ex Gordon induces apoptotic cell death in hepatocellular carcinoma HepG2 cell via intrinsic pathway by PARP and Topoisomerase I suppression.

Pinus kesiya Royle ex Gordon (PK), widely found in Southeast Asia, has been traditionally used for the treatment of several illnesses. Our previous studies showed that PK was highly cytotoxicity against liver cancer cells. The detailed mechanism of anticancer action of 50% hydro-ethanolic extract of PK's twig was, therefore, investigated in hepatocellular carcinoma HepG2 cells. Cytotoxicity of PK was determined by using NR assay, followed by determination of the mode of cell death by flow cytometry. The apoptosis-inducing effect was determined based on caspases activity, mitochondria membrane potential change, and expression of proteins related to apoptosis by western blot. The biomolecular alteration in the PK-treated HepG2 cells was investigated by FTIR microspectroscopy. Inhibition of topoisomerase I enzyme was determined by using DNA relaxation assay. Results showed that PK displayed high selective cytotoxicity and induced apoptosis against HepG2. FTIR microspectroscopy indicated that PK altered major biomolecules in HepG2 different from melphalan (a positive control), indicating a different mechanism of anticancer action. PK induced apoptotic cell death through the intrinsic pathway by increasing caspases 9 and 3/7 activity, increasing Bax, and decreasing Bcl-2 expression leading to mitochondrial membrane potential changes. PK also inhibited Top I and PARP activity that triggered an intrinsic apoptotic pathway. The phytochemical test presented terpenoids (i.e., α-pinene confirmed by GC-MS), alkaloids, steroids, xanthone, reducing sugar, and saponin. α-Pinene exhibited low cytotoxicity against HepG2, therefore, several terpene derivatives may work synergistically for inducing apoptosis. Our data demonstrated that PK has the potential for further study with chemotherapeutic purposes.

Evaluation of Melanoma (SK-MEL-2) Cell Growth between Three-Dimensional (3D) and Two-Dimensional (2D) Cell Cultures with Fourier Transform Infrared (FTIR) Microspectroscopy.

Fourier transform infrared (FTIR) microspectroscopy was used to evaluate the growth of human melanoma cells (SK-MEL-2) in two-dimensional (2D) versus three-dimensional (3D) spheroid culture systems. FTIR microspectroscopy, coupled with multivariate analysis, could be used to monitor the variability of spheroid morphologies prepared from different cell densities. The characteristic shift in absorbance bands of the 2D cells were different from the spectra of cells from 3D spheroids. FTIR microspectroscopy can also be used to monitor cell death similar to fluorescence cell staining in 3D spheroids. A change in the secondary structure of protein was observed in cells from the 3D spheroid versus the 2D culture system. FTIR microspectroscopy can detect specific alterations in the biological components inside the spheroid, which cannot be detected using fluorescence cell death staining. In the cells from 3D spheroids, the respective lipid, DNA, and RNA region content represent specific markers directly proportional to the spheroid size and central area of necrotic cell death, which can be confirmed using unsupervised PCA and hierarchical cluster analysis. FTIR microspectroscopy could be used as an alternative tool for spheroid cell culture discrimination, and validation of the usual biochemical technique.

Tyrosine-Chlorambucil Conjugates Facilitate Cellular Uptake through L-Type Amino Acid Transporter 1 (LAT1) in Human Breast Cancer Cell Line MCF-7.

l-type amino acid transporter 1 (LAT1) is an amino acid transporter that is overexpressed in several types of cancer and, thus, it can be a potential target for chemotherapy. The objectives of this study were to (a) synthesize LAT1-targeted chlorambucil derivatives and (b) evaluate their LAT1-mediated cellular uptake as well as antiproliferative activity in vitro in the human breast cancer MCF-7 cell line. Chlorambucil was conjugated to l-tyrosine-an endogenous LAT1 substrate-via either ester or amide linkage (compounds 1 and 2, respectively). While chlorambucil itself did not bind to LAT1, its derivatives 1 and 2 bound to LAT1 with a similar affinity as with l-tyrosine and their respective cellular uptake was significantly higher than that of chlorambucil in MCF-7. The results of our cellular uptake study are indicative of antiproliferative activity, as a higher intracellular uptake of chlorambucil derivatives resulted in greater cytotoxicity than chlorambucil by itself. LAT1 thus contributes to intracellular uptake of chlorambucil derivatives and, therefore, increases antiproliferative activity. The understanding gained from our research can be used in the development of LAT1-targeted anticancer drugs and prodrugs for site-selective and enhanced chemotherapeutic activity.