Cytotoxic Activity, and Molecular Docking of Indole Alkaloid Voacangine and Bisindole Alkaloid Vobtusine, Vobtusine Lactone from the Indonesian Plant: Voacanga foetida (Blume) Rolfe

ABSTRACT The purpose of this study was to isolate and test its cytotoxic activity starting from extract fraction and its isolate compound, then carried out molecular docking to confirm the potential biological activity of ligands (vocangine, vobtusine, and vobtusine lactone) against inhibition proteins (Bcl-2, Bcl-xL, and Mcl-1), activation protein Bax and activation of apoptotic execution protein Caspase-3. This research is an experimental quantitative study using column chromatography and HPLC methods in the isolation process, MTT assay in determining the cytotoxic activity, and molecular docking in determining the prediction of apoptotic mechanism. The cytotoxic activity of VFB-DA, VFB-DB, VFB-BuOH, VFB-DB4 fractions, voacangine compounds, vobtusine are very strong. while vobtusine lactone is moderate cytotoxic activity. The docking score for voacangine, vobtusine, and vobtusine lactone compounds against Bcl-2 is -9.93; -10.07; -9.03 kcal/mol, against Bcl-xl is -9.77; -11.69; -9.76 kcal/mol, against Mcl-1 is -10.70; -10.77; -9.53 kcal/mol, and for Bax is -8.99; -6.87; -6.99 kcal/mol, as well as against caspase3 is -12.05; -12.21; -12.02 kcal/mol. The cytotoxic activity of voacangine, vobtusine, and vobtusine lactone compounds is thought to cause cell death by suppressing Bcl-2 activity; Bcl-xl; and Mcl-1, increased Bax activity and increased caspase3 activity. Key words: Voacanga foetida, in vitro, in silico

Effect of different concentrations of lithium chloride on p-GSK-3β content in a model of ischemic stroke

Введение. В современном мире проблема инсультов постепенно выходит на лидирующие позиции. Отсутствие эффективных медикаментозных методов коррекции острого нарушения мозгового кровообращения приводит к необходимости поиска новых препаратов с нейропротекторным потенциалом, способных если не предотвратить, то значимо минимизировать последствия и тяжесть ишемического инсульта. Цель исследования - оценка влияния различных доз хлорида лития на фосфорилирование GSK-3β и выживаемость животных на модели ишемического инсульта. Методика. В исследовании были использованы беспородные крысы - самцы, разделённые на 5 групп: ложнооперированные (n=9), контрольная группа (ишемический инсульт с введением раствора NaCl 0,9% в объеме, эквивалентном вводимым лекарственным средствам в других группах, n=5), и группы с введением хлорида лития в дозах 4,2 мг/кг (n=5), 21 мг/кг (n=5) и 63 мг/кг (n=5). Ишемический инсульт моделировали по методу Лонга. По истечении 7 сут от начала эксперимента животные подвергались гуманной эвтаназии с извлечением головного мозга и дальнейшим определением уровня фосфорилированной формы GSK-3β (p-GSK-3β) методом вестерн-блоттинга. Нейропротекторный эффект солей лития реализуется благодаря прямому ингибированию ключевой киназы аптотического механизма клеточной сигнализации - гликоген-синтазы киназы-3β (GSK-3β) с переводом её в фосфорилированую форму (p-GSK-3β). На 7-е сут также был проведен анализ показателей летальности в группах. Для множественных сравнений рассчитывали критический уровень значимости при использовании поправки Бонферрони. Результат. Хлорид лития в дозе 4,2 мг/кг оказывал минимальное влияние как на уровень p-GSK-3β (p=0,8), так и на летальность по отношению к контрольной группе (p>0,017). Доза 21 мг/кг, в свою очередь, значимо повышала уровень p-GSK-3β (p=0,008), но не снижала летальность (p>0,017) по отношению к группе контроля. При использовании дозировки 63 мг/кг уровень p-GSK-3β был максимально приближен к группе ложнооперированных животных (p=0,007), а летальность на 7 сут была значимо ниже (p>0,017). Заключение. Хлорид лития обладает отчётливым дозозависимым нейропротекторным эффектом. Нейропротекторный эффект солей лития реализуется благодаря прямому ингибированию ключевой киназы аптотического механизма клеточной сигнализации - гликоген-синтазы киназы-3β (GSK-3β) с переводом её в фосфорилированую форму (p-GSK-3β) Реализация нейропротекторного эффекта данного препарата потенциально способна улучшить прогнозы течения ишемического инсульта. Background. Ischemic stroke is becoming a major medical concern worldwide. Reasons for this include the aging population, which experiences an increasing frequency of cardiovascular problems. Additionally, social factors, e.g., smoking, fatigue, substance abuse, lead to strokes in young and middle-aged people. The lack of effective medical methods for correcting acute cerebral circulatory disorders underscores the need for new drugs whose neuroprotective potential can prevent or significantly minimize the consequences and severity of ischemic stroke. Aim. To evaluate the effect of different doses of lithium chloride on GSK-3ß phosphorylation and on animal survival in a model of ischemic stroke. Methods. 29 male rats were divided into five groups: Sham-operated (n=9); control, ischemic stroke with administration of a volume of 0.9% NaCl solution equivalent to the volume of the administered drugs in other groups (n=5); and groups with administration of lithium chloride at doses of 4.2 mg/kg (n=5), 21 mg/kg (n=5), and 63 mg/kg (n=5). Ischemic stroke was produced by the Long method. After 7 days, the animals were subjected to humane euthanasia. The brain was excised, and the phosphorylated form of GSK-3β (p-GSK-3β) was measured by Western blotting. The neuroprotective effect of lithium salts occurs due to a direct inhibition of the key kinase of the apoptotic mechanism of cell signaling, glycogen-synthase kinase (GSK-3β), that is transformed into a phosphorylated form. Also, the group mortality rates were analyzed on day 7. For multiple comparisons, a critical level of significance was calculated using the Bonferroni correction. Results. Lithium chloride, 4.2 mg/kg, had a minimal effect on both p-GSK-3ß (p=0.8) and mortality compared to the control group (p>0.017). A dose of 21 mg/kg significantly increased p-GSK-3ß (p=0.008), but did not reduce mortality (p>0.017), relative to the control group. At a dose of 63 mg/kg, p-GSK-3ß was similar to that of the sham operated animals (p=0.007), and the mortality on day 7 was significantly lower (p>0.017). Conclusion. Lithium chloride produces a dose-dependent, neuroprotective effect. This protective effect occurs due to a direct inhibition of the key kinase of the apoptotic mechanism of cell signaling, glycogen-synthase kinase (GSK-3β), that is transformed into a phosphorylated form. This neuroprotection is potentially able to improve the prognosis of ischemic stroke.

Curcumin as an Anti-Proliferative Agent in Breast Cancer through RASSF1A, Bax, and Caspase-3 Protein

Introduction: Curcumin is a polyphenol that has pharmacological activity that can inhibit tumor cell growth and induce apoptosis through various mechanisms. However, the specific mechanism of curcumin cytotoxicity remains controversial because of many anti- and pro-apoptotic signaling pathways in various cell types. This study aims to examine the relationship among curcumin on RASSF1A, Bax protein levels, and caspase-3 activity in supporting the apoptotic mechanism in CSA03 breast cancer cells. Method: Curcumin administration to cancer cells is based on differences in dosage with 24-hour incubation. Cytotoxicity after curcumin administration was determined using MTS. RASSF1A and Bax protein levels were tested through ELISA. Caspase-3 activity was used to determine apoptosis and was tested using flow cytometry. Results: The results indicated that curcumin had a cytotoxicity effect of 40.85 µg/mL. At concentrations of 40 µg/mL and 50 µg/mL, curcumin increases levels of protein RASSF1A (∆ = 26.53% and 47.35%, respectively), Bax (∆ = 48.79% and 386.15%), and caspase-3 (∆ = 1,678.51% and 1,871.889%) significantly. Conclusions: Curcumin exhibits anti-proliferative activity and apoptotic (Caspase-3) effects through activation of RASSF1A and Bax.

Antitumor Activity and Mechanism of Action of Hormonotoxin, an LHRH Analog Conjugated to Dermaseptin-B2, a Multifunctional Antimicrobial Peptide

Prostate cancer is the most common cancer in men. For patients with advanced or metastatic prostate cancer, available treatments can slow down its progression but cannot cure it. The development of innovative drugs resulting from the exploration of biodiversity could open new therapeutic alternatives. Dermaseptin-B2, a natural multifunctional antimicrobial peptide isolated from Amazonian frog skin, has been reported to possess antitumor activity. To improve its pharmacological properties and to decrease its peripheral toxicity and lethality we developed a hormonotoxin molecule composed of dermaseptin-B2 combined with d-Lys6-LHRH to target the LHRH receptor. This hormonotoxin has a significant antiproliferative effect on the PC3 tumor cell line, with an IC50 value close to that of dermaseptin-B2. Its antitumor activity has been confirmed in vivo in a xenograft mouse model with PC3 tumors and appears to be better tolerated than dermaseptin-B2. Biophysical experiments showed that the addition of LHRH to dermaseptin-B2 did not alter its secondary structure or biological activity. The combination of different experimental approaches indicated that this hormonotoxin induces cell death by an apoptotic mechanism instead of necrosis, as observed for dermaseptin-B2. These results could explain the lower toxicity observed for this hormonotoxin compared to dermaseptin-B2 and may represent a promising targeting approach for cancer therapy.

Davidone C Induces the Death of Hepatocellular Carcinoma Cells by Promoting Apoptosis and Autophagy

Davidone C is a newly discovered flavonoid compound purified from the ethyl acetate-soluble fraction of Sophora davidii (Franch.) Skeels. This study explored the anti-tumor activity of davidone C on hepatocellular carcinoma HepG2 and Bel-7402 cells and its mechanism through MTT method, morphological observation, flow cytometry and Western blotting. The results showed that davidone C significantly inhibited the proliferation of HepG2 and Bel-7402 cells in a time- and dose-dependent manner. The morphological changes of apoptotic cells can be observed under an inverted microscope, such as cell floating, chromosome condensation, apoptotic bodies, and other phenomena. The expressions of Bax, cleaved caspase-9, cleaved caspase-3 and cleaved PARP increased with the increase of dosage while Bcl-2 decreased, suggesting that the apoptotic mechanism might be related to the mitochondrial apoptotic pathway. Moreover, davidone C administration can down-regulate the expression of Grp78, and simultaneously up-regulate the expression of caspase-7 and caspase-12, indicating that the apoptotic mechanism might be related to the ERS pathway. In addition, davidone C can down-regulate the expression of p62, and simultaneously up-regulate the expression of LC3-I and LC3-II with a quantitative dependence, suggesting that the mechanism of apoptosis may be related to the autophagy signal pathway. All these results showed davidone C has potential effects on hepatocellular carcinoma.