Celastrol inhibit the proliferation, invasion and migration of human cervical HeLa cancer cells through down‐regulation of MMP‐2 and MMP‐9

The present study evaluated the anticancer potential of celastrol through down‐regulation of matrix metalloproteinase‐2 (MMP‐2) and MMP‐9. HeLa cells were incubated with different concentrations of celastrol (1, 10 and 100 µM) for 48h. Doxorubicin was used as a reference drug. Cancer cell migration, apoptosis, cell viability and mitochondrial fragmentation were evaluated following celastrol treatment. In addition, the expression level of MMP‐2, MMP‐9 and caspase‐3 was evaluated following celastrol treatment. HeLa cell viability was 94.1 ± 7, 53.4 ± 4 and 36.3 ± 2% at 1‐100 µM of celastrol, respectively. Apoptotic cell numbers were increased, and inhibition of larger wounds in cancer cells was observed following celastrol treatment. Celastrol‐treated cells showed condensed nuclei and clumped mitochondria. Reduced expression of MMP‐2 and MMP‐9 and increased expression of caspase‐3 were observed following celastrol treatment. Based on the experimental results, we are concluding that the celastrol was effective against HeLa cervical cancer cells.


Funding information
None.

Abstract
The present study evaluated the anticancer potential of celastrol through downregulation of matrix metalloproteinase-2 (MMP-2) and MMP-9. HeLa cells were incubated with different concentrations of celastrol (1, 10 and 100 µM) for 48h.
Doxorubicin was used as a reference drug. Cancer cell migration, apoptosis, cell viability and mitochondrial fragmentation were evaluated following celastrol treatment.
In addition, the expression level of MMP-2, MMP-9 and caspase-3 was evaluated following celastrol treatment. HeLa cell viability was 94.1 ± 7, 53.4 ± 4 and 36.3 ± 2% at 1-100 µM of celastrol, respectively. Apoptotic cell numbers were increased, and inhibition of larger wounds in cancer cells was observed following celastrol treatment. Celastrol-treated cells showed condensed nuclei and clumped mitochondria.
Reduced expression of MMP-2 and MMP-9 and increased expression of caspase-3 were observed following celastrol treatment. Based on the experimental results, we are concluding that the celastrol was effective against HeLa cervical cancer cells.

K E Y W O R D S
apoptosis, celastrol, cervical cancer, mitochondrial fragmentation, proliferation 2 | MATERIAL S AND ME THODS

| Materials
Celastrol (C0869 Shangai, China), foetal bovine serum (FBS), antibiotics and RPMI medium were obtained from Sigma-Aldrich (Shanghai, China). Primary antibodies of MMP-2 (ab97779), MMP-9 (ab38898) and caspase-3 (a190437) were purchased from Abcam, Cambridge, UK. Acridine orange (AO, A9231) and ethidium bromide (EB, E8751) were obtained from Sigma-Aldrich (Shanghai, China). The growth-inhibiting effects of celastrol on human HeLa cervical cancer cells and primary cervical epithelial cells were investigated by Sulphorhodamine B assay. 7 Wound healing and migration assays were assessed using a previously reported method. 7 AO/EB staining is commonly used for evaluating apoptosis. 7 Mitochondrial fragmentation of human cervical cells was assessed using a previously reported method. 8 The mRNA expression was determined according to a previously reported method. 7 The specific primers used for the amplification of MMP-2, MMP-9 and caspase-3 are shown in Table S1. Western blot analysis was used to evaluate the protein levels in the cancer cells according to a previously described method. 7 Immunofluorescence was performed according to previously reported method. 7

| Statistical analyses
Experimental values are given as the means with standard error of the mean (SEM). The differences between the control and celastroltreated groups were evaluated using Student's t test and analysis of variance (ANOVA). P <.05 was taken as significant.

| D ISCUSS I ON
Cervical cancer is the frequently occurring cancer type in female population worldwide. 1 Few plant-derived drugs with good sideeffect profiles are available for treating cervical cancer. 2 Researchers have reported that cervical cancer develops due to cultural and lifestyle factors, lack of access to health care and exposure to human papillomaviruses. 3 Researchers have reported that the plant-derived natural agents induce apoptosis and exert anticancer effects. 4 Natural products such as celastrol were effective against ovarian cancers. 9 Bufu et al 10 reported that the anti-proliferative effect of celastrol against colorectal cancer was exerted through inhibition of MMP-3 and MMP-7, and migration. In this study, we evaluated the anticancer potential of celastrol through down-regulation of matrix metalloproteinase-2 (MMP-2) and MMP-9. Our results showed that the celastrol decreased cervical cancer cell proliferation, and the expression of MMP-2 and MMP-9, indicating reduced cell migration.
Our results agreed with Hu et al, 5 who reported that celastrol inhibited cervical cancer cell proliferation and migration.
Celastrol inhibited cervical cancer cell viability than doxorubicin. Epithelial cell viability was not altered significantly, which indicates that celastrol does not exert cytotoxic effects on primary cervical epithelial cells. However, 1 mM of celastrol reduced cervical epithelial cell viability. HeLa cells treated with celastrol increased apoptosis, and apoptosis is a programmed death cancerous cells. 11 Celastrol exhibits anticancer activity through inhibition of cell proliferation and induction of apoptosis. 9 Bioactive compounds mediated inhibition of MMP-2 and MMP-9 expression leads to inhibition of cancer cell metastasis. 12 13 Caspase-3 is an important regulator of the apoptotic response during tumorigenesis. 14 Anticancer effects of compounds have been reported through the up-regulation of caspase-3 in cancer cells. 15 In this study of celastrol, caspase-3 expression was increased in HeLa cells, which was degree comparable to doxorubicin.
Researchers have reported the celastrol inhibited the expression of MMP-2 and MMP-9, and which leads anticancer activity in ovarian cancer cells. Celastrol exhibits anticancer activity by inducing cytotoxicity, apoptosis, mitochondrial fragmentation and the expression of caspase-3. Celastrol inhibits cervical cancer cell migration and MMP-2/9 expression. Based on the experimental results, we are concluding that the celastrol was effective against HeLa cervical cancer cells.

ACK N OWLED G EM ENT
None.

CO N FLI C T O F I NTE R E S T
All authors declare that they have no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data and material during the current study are available from the corresponding author on reasonable request.