Note: p38 inhibitor inhibits the apoptosis of cowanin-treated human colorectal adenocarcinoma cells

Note: p38 inhibitor inhibits the apoptosis of cowanin-treated human colorectal adenocarcinoma cells

(doi: 10.3892/ijo.2018.4353)

Cowanin is pure cmp. from Garcinia cowa (Chamuay);

Crude extract from tree;

1. antitimor activity

2. inflmmation induction

3. antibacterial activity

4. anti-inflammatory activity

5. antimalarial activity

Purpose of this study,

1. effects of cowanin on apoptosis induction

2. effect on apoptosis-related protein kinase

3.mitogen-activated protein kinase

Target cell line -- LoVo human colorectal cancer cell line

Assay test;

1. MTT assay (viability assay)

2. Nuclear morphological change by Hoe staining

3. Mitochondrial membrane potential by JC-1 staining

Cowanin inhibits cell proliferation and induced cell dead via apoptosis pw.

Cowanin induce all apoptotic related molecules through MAPK and Akt signaling pw. Therefore, the author suggested that cowanin might be a good candidate to treat colorectal cancer.

Two pathways to induce apoptosis;

1. intrinsic mitochrodria pw

2. extrinsic death receptor pw

Cowanin is active against MCF-7 breast cancer cells. In addition, cowanin extracted from leaves was evaluated for cytotoxicity against NCl-H187, KB and MCF-7 cell lines with IC50 values of 3.87-8.58, 6.43‑15.43 and 10.59‑21.38 μg/ml,

Gap;

cowanin on colorectal cancer has not yet been reported.

Purpose;

1.investigate whether cowanin induce apoptosis

2.investigate whether cowanin inhibit growth in CRC cell line

There are several assays to answer the questions;

1. MTT assay

2. Nucleus morphology - cell shrinkage, mb blebbing, and chromatin condensation

3. At the molecular level, looking at the gene expression of caspase, Bcl-2 family, MAPK and Akt signaling pw.

Cowanin is used;

20ug/ml

15ug/ml

10ug/ml

5ug/ml

Final DMSO 0.5%

incubate 3,6,9,12 and 24 hrs.

Normal cell line; COS-7

Check whether the lost of viability during treating with cowanin caused by apoptotic induction.

mitochondrial depolarization is indicated by a decrease in the red/green fluorescence intensity ratio.

p38 inhibitor -- SB203580

MEK1/2 inhibitor -- U0126

During apoptosis, DNA is degraded by cellular endonucleases resulting in less DNA compared with healthy cells, thus representing the sub‑G1 peak in flow cytometry analysis.

the caspase cascade, caspase‑9 causes the activation of caspase‑7, which then cleaves vital substrates, such as PARP, that play a vital role in DNA repair. cowanin induced apoptosis via the mitochondrial pathway.

the present study demonstrated for the first time that cowanin treatment inhibited cell proliferation and

induced apoptosis in colorectal LoVo cells, via intrinsic or mitochondria pathway activation. In addition, cowanin downregulated ERK and Akt signaling and upregulated p38 MAPK signaling.

x