Anticancer Activity of Cissus quadrangularis: An in vitro 2D Model Based Study

Cissus quadrangularis (CQ) is a perennial rambling shrub of the grape family commonly known as “Hadjora” (in Hindi) probably native to India or Sri Lanka. It is one of the valuable medicines in the Indian Traditional Systems of Medicine because of the presence of several bioactive compounds. However, in the present study we have checked its anticancer activity along with its safety profile on normal skin cells. Apart from this we have generated the spheroid HeLa culture in vitro model for analyzing the CQ extract response on the growth of HeLa tumoroid. From the present findings we have observed that the CQ selectively induces cytotoxicity, ROS liberation and G1 phase cell cycle arrest only in HeLa cancer cells without affecting the normal skin cells at similar dose. CQ also significantly inhibits the growth of tumoroid and finally leads to cell death as revealed by phase contrast microscopy. Therefore, it can be concluded from the present findings that CQ extract shows targeted anticancer activity, though the mechanism of action in support of its exhibited activity needs to be further explored

Cissus quadrangularis Linn. Stem Ethanolic Extract Liberates Reactive Oxygen Species and Induces Mitochondria Mediated Apoptosis in KB Cells

Background: Cissus quadrangularis Linn. (CQ) commonly known as Hadjod (Family: Vitaceae) is usually distributed in India and Sri Lanka and contains several bioactive compounds responsible for various metabolic and physiologic effects. Objective: In this study, the biological effects of CQ ethanolic extract were evaluated by in vitro and supported by in silico analysis on KB oral epidermoid cancer cell line. Materials and methods: Anti-cancer potential of ethanolic extract of CQ stem against KB oral epidermoid cancer cells was evaluated in terms of morphological analysis, nuclei staining, liberation of reactive oxygen species (ROS), cell cycle arrest, mitochondrial membrane potential (MMP) and p53 and Bcl-2 protein expression which reveal the induction of apoptosis along with supporting in silico analysis. Results: Ethanolic extract of CQ stem contains various bioactive compounds responsible for cancer cell morphological alterations, liberation of ROS, G1 phase cell cycle arrest and decreased MMP along with up-regulation of p53 and down-regulation of Bcl-2. By employing in silico approach, we have also postulated that the CQ extract active constituents sequester Bcl-2 with higher affinity as compared to p53, which may be the reason for induction of growth arrest and apoptosis in KB cells. Conclusion: Our data indicate that the CQ extract has a remarkable apoptotic effect that suggests that it could be a viable treatment option for specific types of cancers. Summary: Cissus quadrangularis stem ethanolic extract induces apoptosis and cell cycle arrest at G1 phaseIt liberates (ROS) and mitochondria mediated apoptosisIt upregulates p53 and down-regulates Bcl-2 protein expressionIn silico studies indicates that the active constituents of CQ binds Bcl-2 with higher affinity as compared to p53

Anticancer Activity of Cissus quadrangularis: An in vitro 2D Model Based Study

Cissus quadrangularis (CQ) is a perennial rambling shrub of the grape family commonly known a

Rohitukine affected the apoptosis-associated protein levels in A549 cells.

<p><b>Cells were treated with Rohitukine at 30</b> μ<b>M for 24hrs, and then the total proteins were prepared and determined as described in methods.</b> (a) The levels of proteins expression of p53 and Bcl-2 (b) proteins expression of caspase9 were estimated by Western blotting. Band intensities were calculated by densitometry and change in protein expression after Rohitukine treatment was calculated with respect to controls and expressed as fold change in graph. (c), (d) & (e) densitometry for p53, Bcl-2, and caspase9 blot respectively. Results were normalized to β-actin. The data are represented as means ±SD of three independent experiments (** <i>P</i> < 0.01 versus control).</p

Rohitukine causes DNA damage and induction of apoptosis.

<p>(a) A.O. staining (b) Graphical representation for fluorescence intensity of apoptotic death of the Yeast cells as quantified using Image J software ***p < 0.001 (c) DNA damage revealed by Nuc Blue Live Cell Stain (d) Graphical representation for fluorescence intensity of nucleic acid of the yeast cells as quantified using Image J software ***p < 0.001.</p

Rohitukine promoted ROS production and loss Mitochondrial content in gene knockout strains of yeast.

<p>(a) DCFDA staining (b) Graphical representation of Relative formation of reactive oxygen species (ROS) measured by H2DCFDA staining in WT and gene knockout strains of yeast as quantified using Image J software ***p < 0.001.(c) Mitotracker Deep Red staining (d) Graphical representation for fluorescence intensity of mitochondrial content of the budding yeast as quantified using Image J software ***p <0.001.</p

(a) RT-PCR analysis of <i>Slt2</i> and <i>Hog1</i> gene in budding yeast after drug treatment (i:Untreated control, ii: drug treated) (b) The expression of Slt2 and Hog1 mRNA, expressed as the ratio of densitometric measurement of the sample to the corresponding internal control (β-actin) (i: Untreated control, ii: drug treated) (c) Docking studies of Rohitukine with human two different type of member of MAPK pathway. (i) p38 (Hog1 in <i>S</i>. <i>cerevisiae</i>) and (ii) ERK5 (Slt2 in <i>S</i>. <i>cerevisiae</i>).

<p>(a) RT-PCR analysis of <i>Slt2</i> and <i>Hog1</i> gene in budding yeast after drug treatment (i:Untreated control, ii: drug treated) (b) The expression of Slt2 and Hog1 mRNA, expressed as the ratio of densitometric measurement of the sample to the corresponding internal control (β-actin) (i: Untreated control, ii: drug treated) (c) Docking studies of Rohitukine with human two different type of member of MAPK pathway. (i) p38 (Hog1 in <i>S</i>. <i>cerevisiae</i>) and (ii) ERK5 (Slt2 in <i>S</i>. <i>cerevisiae</i>).</p

Hypersensitivity of ΔSlt2 and ΔHog1 strains to Rohitukine.

<p>(a) Yeast cells viability at different concentration of Rohitukine after 24 hrs of drug treatment, 5-fold serial dilutions from exponentially growing cultures of WT, ΔSlt2 and ΔHog1 strains were spotted onto YPD medium containing 40 μg/ml, 80 μg/ml and 100 μg/ml of drug. (b)The percentage of surviving cells relative to untreated controls.</p

Sequence of primers used.

<p>Sequence of primers used.</p

Rohitukine affected the percentage of viable and induced ROS in A549 cells.

<p>(a) Cell viability was determined using the MTT assay. Cells (1 × 10⁴ cells/well; 96 well plates) were plated in DMEM F12 medium + 10% fetal bovine serum (FBS) with 0, 10, 20, 30, 40, 50 and 60 μM for 24 hrs, (b) ROS generation was assessed in terms of relative fluorescence units using 10 mM DCFH-DA in A549 cells after 24 hrs exposure to Rohitukine in black-bottomed 96-well plates and (c) Fluorescence micrographs of ROS generation at 20μM, 30μM, and 40μM Rohitukine concentrations in lung cancer cells obtained at 20X objective after 24 hrs of treatment. The results are represented as means ±S.D of three independent experiments. Nonsignificant (ns), * <i>P</i> < 0.05, ** <i>P</i> < 0.01, *** <i>P</i> < 0.001 versus 0 μM.</p