Cytotoxic evaluation of Leptospermum javanicum and Baeckea frutescens along with the anti-cancer activities of the active isolate, betulinic acid / Suerialoasan Navanesan

The usage of natural products as precursors for pharmacologically significant outputs has longed yielded positive outcomes. Along these lines, this study chose to look into Leptospermum javanicum and Baeckea frutescens, both found in mountainous region in Peninsular Malaysia and have limited biological investigations. The aim of the present study was to assess the cytotoxicity of both plants against lung cancer cells, followed by evaluating the anti-cancer and anti-metastatic properties of the active extracts and their compound. In addition to this, the protein expression of the active compound was evaluated to obtain a better understanding into the mechanism of action. The crude methanol extracts of both plants showed no signs of acute oral toxicity when tested on Sprague-Dawley rats. The cytotoxic potentials of the extraction yields (methanol, hexane, ethyl acetate and water extracts as wells as a semi pure fraction, LF1 from L. javanicum) were evaluated against two human non-small cell lung carcinoma cell lines (A549 and NCI-H1299) using the MTT assay, with LF1 being the most promising. LF1 treatment resulted in the manifestation of a sub-G1 region in the cell cycle analysis while also causing presences of apoptotic morphologies in cells stained with acridine orange and ethidium bromide (AO/EB). Treatment with LF1 resulted in an apoptotic population in cells that were evaluated using the Annexin V/ propidium iodide assay. Blockage of cell cycle progression was also observed in LF1-treated cells. Following this, LF1 underwent isolation and purification to yield a white powder which was identified as Betulinic acid (BA) via NMR, LCMS and IR spectroscopy. The isolate, BA, which produced an encouraging cytotoxic effect against A549 and NCI-H1299 cells through the MTT assay, was further assessed with AO/EB staining, TUNEL assay, sub-G1 population quantification as well as activated caspase-3 detection. The data pointed towards the induction of apoptosis as a result of increasing concentrations of BA, regardless of the p53 status in both cell lines. Treatment with BA also prevented an effective attachment of the invasive A549 cells onto a new culture surface, in addition to diminishing the migratory and invasive potential of treated cells across an uncoated porous membrane and a Matrigel-coated membrane, respectively. Further investigation through the ELISA detection method and gelatin zymography showed an adverse effect to production of matrix metalloproteinase-2 (MMP-2). Protein expression of BA-treated cells showed decreased levels of p-Akt, ERK1/2 and cyclin D1 followed by a slight increase in p21 and p27 expression, all of which is believed to have contributed to the G0/G1 arrest. In addition to this, alteration in NF-κB, IκB-α, STAT3 and JNK phosphorylation due to treatment with BA could have been a contributing factor to its apoptotic and anti-metastatic properties. Taken together, the results showed that the L. javanicum isolate, BA, possess anti-cancer effects through induction of apoptosis, restriction of cell cycle progression and anti-metastatic effects on human lung cancer cells. The ability of BA to not only eradicate but control the spread of lung cancer cells makes it a promising lead compound in the treatment of lung cancer

Leptospermum flavescens Constituent-LF1 Causes Cell Death through the Induction of Cell Cycle Arrest and Apoptosis in Human Lung Carcinoma Cells.

Leptospermum flavescens Sm. (Myrtaceae), locally known as 'Senna makki' is a smallish tree that is widespread and recorded to naturally occur in the montane regions above 900 m a.s.l from Burma to Australia. Although the species is recorded to be used traditionally to treat various ailments, there is limited data on biological and chemical investigations of L. flavescens. The aim of the present study was to investigate and understand the ability of L. flavescens in inducing cell death in lung cancer cells. The cytotoxic potentials of the extraction yields (methanol, hexane, ethyl acetate and water extracts as wells as a semi pure fraction, LF1) were evaluated against two human non-small cell lung carcinoma cell lines (A549 and NCI-H1299) using the MTT assay. LF1 showed the greatest cytotoxic effect against both cell lines with IC50 values of 7.12 ± 0.07 and 9.62 ± 0.50 μg/ml respectively. LF1 treated cells showed a sub-G1 region in the cell cycle analysis and also caused the presence of apoptotic morphologies in cells stained with acridine orange and ethidium bromide. Treatment with LF1 manifested an apoptotic population in cells that were evaluated using the Annexin V/ propidium iodide assay. Increasing dosage of LF1 caused a rise in the presence of activated caspase-3 enzymes in treated cells. Blockage of cell cycle progression was also observed in LF1-treated cells. These findings suggest that LF1 induces apoptosis and cell cycle arrest in treated lung cancer cells. Further studies are being conducted to isolate and identify the active compound as well to better understand the mechanism involved in inducing cell death

Effects of exposure to LF1 on the externalization of PS in NCI-H1299 at different time points (Time—dependent).

<p>Cells were treated with 15 μg/ml of LF1 for 24 hours (A), 48 hours (B) and 72 hours (C). Summary of the results were presented in a bar chart (D). An increase in early apoptotic cells over time that was then followed by an increase in double positively stained cells indicate an apoptotic pathway was taken by LF1 treated cells.</p

Morphological observation of LF1 treated A549 and NCI-H1299 using AO/EB staining at × 400 magnifications.

<p>A549 and NCI-H1299 were treated without (untreated control) and with LF1 at different concentrations. Green arrows indicate early apoptotic cells (chromatin condensation stained green); blue arrows are late apoptotic cells (chromatin condensation stained orange); purple arrows shows membrane blebbing; yellow arrows are for cells which appear shrunken and red arrows indicate loss of membrane shape. The higher the concentration of LF1 used, the more aggressive pathway taken in the induction of death in the cancer cells.</p

Presence of active caspase-3 in LF1 treated A549 and NCI-H1299.

<p>(A) A549 and (B) NCI-H1299 cells were treated with 5, 10 and 20 μg/ml of LF1 for 24 hours. Summary of the results were presented in a bar chart (C). An increase in the presence of cleaved caspase-3 was detected in both cell lines in a dose-dependent manner, indicating an apoptotic pathway was taken by LF1 treated cells.</p

Effects of exposure to LF 1 for 24 hours on the externalization of PS in A549 and NCI-H1299 (Dose—dependent).

<p>Summary of the results for 24 hours treatment with LF1 was presented in a bar chart (A) for A549 and bar chart (B) for NCI-H1299. A significant increase in the early apoptotic and secondary necrotic cell population is observed with the increasing concentration of LF1 in both cell lines.</p

Sub G₁ population of LF1 treated cells.

<p>A549 (A) and NCI-H1299 (B) cells were treated with 5, 7.5 and 10 μg/ml of LF1. The resultant sub-G₁ population show an increase with the increase in concentration of LF1 used.</p

Morphological observation of LF1 treated A549 and NCI-H1299 using AO/EB staining at × 400 magnifications.

<p>A549 and NCI-H1299 were treated without (untreated control) and with LF1 at different concentrations. Green arrows indicate early apoptotic cells (chromatin condensation stained green); blue arrows are late apoptotic cells (chromatin condensation stained orange); purple arrows shows membrane blebbing; yellow arrows are for cells which appear shrunken and red arrows indicate loss of membrane shape. The higher the concentration of LF1 used, the more aggressive pathway taken in the induction of death in the cancer cells.</p

Effects of LF1 on cell cycle distribution in A549 NCI-H1299 cells.

<p>A549 (A) and NCI-H1299 (B) were incubated in absence (control) and presence of LF1 at 5, 7.5 and 10 μg/ml for 24 hours. Summary of results indicate an increase in G₀/G₁ population with increasing dosages of LF1 used.</p

Conolodinines A–D, Aspidosperma–Aspidosperma Bisindole Alkaloids with Antiproliferative Activity from Tabernaemontana corymbosa

Examination of the EtOH extract of the leaves of the Malayan Tabernaemontana corymbosa resulted in the isolation of four new (1-4) and two known bisindole alkaloids (5, 6) of the Aspidosperma-Aspidosperma type. The structures of these alkaloids were determined based on analysis of the spectroscopic data (NMR and HRESIMS). X-ray diffraction analyses of the related bisindole alkaloids conophylline (5) and conophyllinine (6) established the absolute configurations. Treatment of the bisindole alkaloid conophylline (5) with benzeneselenic anhydride gave, in addition to the known bisindole polyervinine (7) previously isolated from another Malayan Tabernaemontana, another bisindole product, 8, an isolable tautomer of 7. X-ray diffraction analyses yielded the absolute configurations of both bisindoles and in addition showed that polyervinine (7) exists primarily as the neutral dione structure. The bisindoles (1-8) and the related conophylline-type bisindoles (9-13) showed pronounced in vitro growth inhibitory activity against an array of human cancer cell lines, including KB, vincristine-resistant KB, PC-3, LNCaP, MCF7, MDA-MB-231, A549, HT-29, and HCT 116 cells, with IC50 values for the active compounds in the 0.01-5 μM range. © 2019 American Chemical Society and American Society of Pharmacognosy