Gallic Acid Enhances the Efficacy of BCR::ABL1 Tyrosine Kinase Inhibitors in Chronic Myeloid Leukemia through Inhibition of Mitochondrial Respiration and Modulation of Oncogenic Signaling Pathways.

While BCR::ABL1 tyrosine kinase inhibitors have transformed the treatment paradigm for chronic myeloid leukemia (CML), disease progression and treatment resistance due to BCR::ABL1-dependent and BCR::ABL1-independent mechanisms remain a therapeutic challenge. Natural compounds derived from plants have significantly contributed to cancer pharmacotherapy. This study investigated the efficacy of an active component of Leea indica, a local medicinal plant, in CML. Using high-performance liquid chromatography-electrospray ionization-mass spectrometry, a chemical constituent from L. indica extract was isolated and identified as gallic acid. Commercially obtained gallic acid was used as a chemical standard. Gallic acid from L. indica inhibited proliferation and induced apoptosis in CML cell lines, as did the chemical standard. Furthermore, gallic acid induced apoptosis and decreased the colony formation of primary CML CD34+ cells. The combination of isolated gallic acid or its chemical standard with BCR::ABL1 tyrosine kinase inhibitors resulted in a significantly greater inhibition of colony formation and cell growth compared to a single drug alone. Mechanistically, CML cells treated with gallic acid exhibited the disruption of multiple oncogenic pathways including ERK/MAPK, FLT3 and JAK/STAT, as well as impaired mitochondrial respiration. Rescue studies showed that gallic acid is significantly less effective in inducing apoptosis in mitochondrial respiration-deficient ρ0 cells compared to wildtype cells, suggesting that the action of gallic acid is largely through the inhibition of mitochondrial respiration. Our findings highlight the therapeutic potential of L. indica in CML and suggest that gallic acid may be a promising lead chemical constituent for further development for CML treatment.

Effects of Leea indica leaf extracts and its phytoconstituents on natural killer cell-mediated cytotoxicity in human ovarian cancer.

BACKGROUND: The rich biodiversity of medicinal plants and their importance as sources of novel therapeutics and lead compounds warrant further research. Despite advances in debulking surgery and chemotherapy, the risks of recurrence of ovarian cancer and resistance to therapy are significant and the clinical outcomes of ovarian cancer remain poor or even incurable. OBJECTIVE: This study aims to investigate the effects of leaf extracts from a medicinal plant Leea indica and its selected phytoconstituents on human ovarian cancer cells and in combination with oxaliplatin and natural killer (NK) cells. METHODS: Fresh, healthy leaves of L. indica were harvested and extracted in 70% methanol by maceration. The crude extract was partitioned with n-hexane, dichloromethane and ethyl acetate. Selected extracts and compounds were analyzed for their effects on cell viability of human ovarian cancer cells, NK cell cytotoxicity, and stress ligands expression for NK cell receptors. They were also evaluated for their effects on TNF-α and IL-1ß production by enzyme-linked immunosorbent assay in lipopolysaccharide-stimulated human U937 macrophages. RESULTS: Leaf extracts of L. indica increased the susceptibility of human ovarian tumor cells to NK cell-mediated cytotoxicity. Treatment of cancer cells with methyl gallate but not gallic acid upregulated the expression of stress ligands. Tumor cells pretreated with combination of methyl gallate and low concentration of oxaliplatin displayed increased levels of stress ligands expression and concomitantly enhanced susceptibility to NK cell-mediated cytolysis. Further, NK cells completely abrogated the growth of methyl gallate-pretreated ovarian cancer cells. The leaf extracts suppressed TNF-α and IL-1ß production in human U937 macrophages. Methyl gallate was more potent than gallic acid in down-regulating these cytokine levels. CONCLUSIONS: We demonstrated for the first time that leaf extracts of L. indica and its phytoconstituent methyl gallate enhanced the susceptibility of ovarian tumor cells to NK cell cytolysis. These results suggest that the combined effect of methyl gallate, oxaliplatin and NK cells in ovarian cancer cells warrants further investigation, for example for refractory ovarian cancer. Our work is a step towards better scientific understanding of the traditional anticancer use of L. indica.

New and efficient purification process for recombinant human insulin produced in Escherichia coli.

A new and efficient purification process for recombinant human insulin production was developed by exploring new resins and optimizing purification steps from E. coli inclusion body washing to insulin polishing. A combined additives inclusion body wash protocol drastically improved efficiency in clarifying ZZ-proinsulin samples. ZZ-proinsulin recovery increased three-fold under optimized solubilization and sulfitolysis incubation temperature and duration. Desalting with Bio-Gel P4 and P6 resulted in higher sample loading and product recovery compared to conventional resins. A higher recovery (96%) and purity (81%) of ZZ-proinsulin were achievable with the Nuvia S cation exchanger for proinsulin purification compared to a reported process using expensive affinity chromatography resin. As the first step for insulin purification, process scale-up is more economical and practical when Nuvia HR-S cation exchanger was used instead of commonly used reversed-phase chromatography. Nuvia HR-S was highly effective in removing ZZ fusion protein (90% removal) after enzymatic cleavage, although ZZ fusion protein has a very close theoretical pI to human insulin, which was supposedly challenging to be removed by cation exchange chromatography. Also, insulin can be eluted at a lower ethanol % using Nuvia HR-S compared to other reported processes and is thus more environmentally sustainable. Recombinant human insulin was obtained with over 98% purity in just a single reversed-phase polishing step, which is comparable to the reference standard. The process workflow presented here can be potentially applied for the development of purification workflow for insulin analogs or other peptide products derived from E. coli inclusion body.Key points• Drastic efficiency improvement for inclusion body wash with combined additives.• High recovery of proinsulin purification with high capacity cation exchange resin.• Effective removal of fusion tag at lower ethanol % with high-resolution resin.

Downstream processing of recombinant human insulin and its analogues production from E. coli inclusion bodies.

The Global Diabetes Compact was launched by the World Health Organization in April 2021 with one of its important goals to increase the accessibility and affordability of life-saving medicine-insulin. The rising prevalence of diabetes worldwide is bound to escalate the demand for recombinant insulin therapeutics, and currently, the majority of recombinant insulin therapeutics are produced from E. coli inclusion bodies. Here, a comprehensive review of downstream processing of recombinant human insulin/analogue production from E. coli inclusion bodies is presented. All the critical aspects of downstream processing, starting from proinsulin recovery from inclusion bodies, inclusion body washing, inclusion body solubilization and oxidative sulfitolysis, cyanogen bromide cleavage, buffer exchange, purification by chromatography, pH precipitation and zinc crystallization methods, proinsulin refolding, enzymatic cleavage, and formulation, are explained in this review. Pertinent examples are summarized and the practical aspects of integrating every procedure into a multimodal purification scheme are critically discussed. In the face of increasing global demand for insulin product, there is a pressing need to develop a more efficient and economical production process. The information presented would be insightful to all the manufacturers and stakeholders for the production of human insulins, insulin analogues or biosimilars, as they strive to make further progresses in therapeutic recombinant insulin development and production.

Identification of Phytoconstituents in Leea indica (Burm. F.) Merr. Leaves by High Performance Liquid Chromatography Micro Time-of-Flight Mass Spectrometry.

Leea indica (Vitaceae) is a Southeast Asian medicinal plant. In this study, an ethyl acetate fraction of L. indica leaves was studied for its phytoconstituents using high-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-microTOF-Q-MS/MS) analysis. A total of 31 compounds of different classes, including benzoic acid derivatives, phenolics, flavonoids, catechins, dihydrochalcones, coumarins, megastigmanes, and oxylipins were identified using LC-MS/MS. Among them, six compounds including gallic acid, methyl gallate, (-)-epigallocatechin-3-O-gallate, myricetin-3-O-rhamnoside, quercetin-3-O-rhamnoside, and 4',6'-dihydroxy-4-methoxydihydrochalcone 2'-O-ß-d-glucopyranoside were isolated and identified by NMR analysis. The LC-MS/MS analysis led to the tentative identification of three novel dihydrochalcones namely 4',6'-dihydroxy-4-methoxydihydrochalcone 2'-O-rutinoside, 4',6'-dihydroxy-4-methoxydihydrochalcone 2'-O-glucosylpentoside and 4',6'-dihydroxy-4-methoxydihydrochalcone 2'-O-(3″-O-galloyl)-ß-d-glucopyranoside. The structural identification of novel dihydrochalcones was based on the basic skeleton of the isolated dihydrochalcone, 4',6'-dihydroxy-4-methoxydihydrochalcone 2'-O-ß-d-glucopyranoside and characteristic LC-MS/MS fragmentation patterns. This is the first comprehensive analysis for the identification of compounds from L. indica using LC-MS. A total 24 compounds including three new dihydrochalcones were identified for the first time from the genus Leea.

Evaluation of anti-proliferative activity of medicinal plants used in Asian Traditional Medicine to treat cancer.

ETHNOPHARMACOLOGICAL RELEVANCE: The extensive biodiversity of plants in Southeast Asia and inadequate research hitherto warrant a continued investigation into medicinal plants. On the basis of a careful review of fresh medicinal plant usage to treat cancer from previous ethnobotanical interviews in Singapore and from the traditional uses of the indigenous plants, fresh leaves of seven locally grown medicinal plant species were evaluated for anti-proliferative activity. AIM OF THE STUDY: To evaluate the anti-proliferative activity of local medicinal plant species Clausena lansium Skeels, Clinacanthus nutans (Burm. f.) Lindau, Leea indica (Burm. f.) Merr., Pereskia bleo (Kunth) DC., Strobilanthes crispus (L.) Blume, Vernonia amygdalina Delile and Vitex trifolia L. MATERIALS AND METHOD: Fresh, healthy and mature leaves of the seven medicinal plants were harvested from various locations in Singapore and Malaysia for Soxhlet, ultrasonication and maceration extractions in three different solvents (water, ethanol and methanol). Cell proliferation assay using water soluble tetrazolium salt (WST-1) assay was performed on twelve human cancer cell lines derived from breast (MDA-MB-231, T47D), cervical (C33A), colon (HCT116), leukemia (U937), liver (HepG2, SNU-182, SNU-449), ovarian (OVCAR-5, PA-1, SK-OV-3) and uterine (MES-SA/DX5) cancer. RESULTS: A total of 37 fresh leaf extracts from seven medicinal plants were evaluated for their anti-tumour activities in twelve human cancer cell lines. Of these, the extracts of C. lansium, L. indica, P. bleo, S. crispus, V. amygdalina and V. trifolia exhibited promising anti-proliferative activity against multiple cancer cell lines. Further investigation of selected promising leaf extracts indicated that maceration methanolic extract of L. indica was most effective overall against majority of the cancer cell lines, with best IC50 values of 31.5 ±â€¯11.4 µg/mL, 37.5 ±â€¯0.7 µg/mL and 43.0 ±â€¯6.2 µg/mL in cervical C33A, liver SNU-449, and ovarian PA-1 cancer cell lines, respectively. CONCLUSION: The results of this study provide new scientific evidence for the traditional use of local medicinal plant species C. lansium, L . indica, P. bleo, S. crispus, V. amygdalina and V. trifolia in cancer treatment. These results highlight the importance of the upkeep of these indigenous plants in modern society and their relevance as resources for drug discovery.

Oxaliplatin regulates expression of stress ligands in ovarian cancer cells and modulates their susceptibility to natural killer cell-mediated cytotoxicity.

Selected cytotoxic chemicals can provoke the immune system to recognize and destroy malignant tumors. Most of the studies on immunogenic cell death are focused on the signals that operate on a series of receptors expressed by dendritic cells to induce tumor antigen-specific T-cell responses. Here, we explored the effects of oxaliplatin, an immunogenic cell death inducer, on the induction of stress ligands and promotion of natural killer (NK) cell-mediated cytotoxicity in human ovarian cancer cells. The results indicated that treatment of tumor cells with oxaliplatin induced the production of type I interferons and chemokines and enhanced the expression of major histocompatibility complex class I-related chains (MIC) A/B, UL16-binding protein (ULBP)-3, CD155 and TNF-related apoptosis-inducing ligand (TRAIL)-R1/R2. Furthermore, oxaliplatin but not cisplatin treatment enhanced susceptibility of ovarian cancer cells to NK cell-mediated cytolysis. In addition, activated NK cells completely abrogated the growth of cancer cells that were pretreated with oxaliplatin. However, cancer cells pretreated with the same concentration of oxaliplatin alone were capable of potentiating regrowth over a period of time. These results suggest an advantage in combining oxaliplatin and NK cell-based therapy in the treatment of ovarian cancer. Further investigation on such potential combination therapy is warranted.

Ethnobotanical survey of usage of fresh medicinal plants in Singapore.

ETHNOPHARMACOLOGICAL RELEVANCE: The use of medicinal plants in human health has been documented since ancient times and they provide a useful source of new therapeutics. In Singapore, despite the accessibility to modern healthcare, there still exist pockets of the population who choose to use locally grown fresh medicinal plants for health promotion and even therapeutic purposes. However to date, there is no published report of first-hand account of their usage in Singapore. As land is scarce and rapidly used for re-development, such important knowledge may be lost if not properly documented in time. This work safeguards the local folk knowledge, and provides information on common and scarcely reported fresh medicinal plants. The objective of this study is to gather information regarding the usage of fresh medicinal plants in Singapore through face-to-face interviews. MATERIALS AND METHODS: Information on demographic data and plant-use methods were collated via face-to-face interviews of 200 fresh medicinal plant users who have used fresh medicinal plants in the last five years. The survey protocol was approved by the National University of Singapore Institutional Review Board and informed consent was obtained from every participant. RESULTS: A total of 414 plants represented by 104 plant species from 44 families were reportedly used by the 200 participants. The five most commonly used plants were Clinacanthus nutans (34 users), Strobilanthes crispus (31 users), Pereskia bleo (25 users), Aloe vera (18 users) and Zingiber officinale (16 users). Leaves were the most commonly used plant part while preparing a decoction was the most common method of preparation. The majority of interviewees used plants for general health purposes and to treat diseases related to the respiratory system and cancer. CONCLUSIONS: Our survey has successfully documented the rich wealth of traditional usage and knowledge on 414 fresh medicinal plants grown in Singapore through face-to-face interviews with 200 users. This study will serve as a useful resource for identifying promising plants for future drug discovery efforts. Scientific evaluations of these medicinal plants are warranted and should be carried out.