2.1. Isolation of Phorbol Esters
The high performance liquid chromatography (HPLC) analysis (
Figure 2) illustrated that the Jatropha meal PEs appeared in four peaks which were labelled as PE1, PE2, PE3 and PE4. Their retention times were similar to those of the PEs reported by Makkar
et al. [
12] and Li
et al. [
13] as described in
Section 3.2. Hass
et al. [
10] have also characterised the PEs of
J. curcas seed and confirmed the presence of the six PEs as shown in
Figure 1. Although the number of the PEs were six but in the present analysis they appeared in four peaks (
Figure 2), this could be possibly due to the similar molecular weight of some of the PEs present in Jatropha meal.
Figure 2.
HPLC chromatogram of the PEs present in Jatropha meal.
Figure 2.
HPLC chromatogram of the PEs present in Jatropha meal.
The concentrations of the isolated PEs used in this study were expressed as equivalents of the standard phorbol-12-myristate 13-acetate (PMA). The total yield of PEs isolated from Jatropha meal was 3 mg PMA equivalent/g dry matter of Jatropha meal. Based on the results observed, the proportions of PE1, PE2, PE3 and PE4 were 57.5, 20.6, 13.9 and 7.8% of the total PEs, respectively. At this stage, due to the lack of information on the biological activities of PEs from Jatropha meal, this study was focused on the cytotoxic properties of the PEs. The PEs were pooled, thus the biological activities observed in this experiment corresponded to the total PEs present in Jatropha meal.
2.2. Proliferation Assay
The anti proliferative activities of isolated PEs and PMA as positive control in MCF-7 and HeLa, are shown in
Figure 3 and
Figure 4 respectively. Isolated PEs and PMA inhibited the cells proliferation in a dose-dependent manner.
Figure 3.
Percentage of cell proliferation inhibition of isolated PEs and PMA on MCF-7 cell line. Values represent mean ± SEM of three replicates.
Figure 3.
Percentage of cell proliferation inhibition of isolated PEs and PMA on MCF-7 cell line. Values represent mean ± SEM of three replicates.
Figure 4.
Percentage cell proliferation inhibition of isolated PEs and PMA on HeLa cell line. Values represent mean ± SEM of three replicates.
Figure 4.
Percentage cell proliferation inhibition of isolated PEs and PMA on HeLa cell line. Values represent mean ± SEM of three replicates.
The IC
50 values presented in
Table 1 show similar concentrations (
p > 0.05) of PEs and PMA to inhibit the proliferation of 50% of the cells for MCF-7 and HeLa cells. The difference in IC
50 values between PMA and PEs for each cell line indicated the possible dissimilarity in the structures of PEs isolated from Jatropha meal.
Table 1.
IC50 concentration of isolated PEs and PMA in MCF-7 and HeLa cell lines.
Table 1.
IC50 concentration of isolated PEs and PMA in MCF-7 and HeLa cell lines.
| IC50 µg/mL |
---|
MCF-7 | HeLa | S.E.M |
---|
PEs | 128.6 | 133.0 | 1.69 |
PMA 1 | 114.7 | 119.6 | 2.16 |
2.3. Microscopic Examination
The results of morphological changes visualized in different cell lines upon treatment with isolated PEs at IC
50 concentration (a–b) after 24 h incubation are presented in
Figure 5. Significant morphological changes, as well as detachment and destruction of cells were observed in both types of cancer cells after 24 h treatment with PEs.
Figure 5.
Morphological changes observed in different cell lines upon treatment with isolated PEs at IC50 concentration (a–b) after 24 h incubation examined by light microscopy at 200× magnification. MCF-7(A,a), HeLa (B,b). The arrows show the apoptotic bodies and destructed cells.
Figure 5.
Morphological changes observed in different cell lines upon treatment with isolated PEs at IC50 concentration (a–b) after 24 h incubation examined by light microscopy at 200× magnification. MCF-7(A,a), HeLa (B,b). The arrows show the apoptotic bodies and destructed cells.
According to these microscopic observations (
Figure 5), the cell damage resembles apoptosis as cell walls were not intact and apoptotic bodies were seen. Both cancer cell lines displayed death upon treatment with the PEs and PMA at IC
50 concentration after 24 h incubation. The present study indicated that PEs isolated from Jatropha meal initially disrupt the cell-substream adhesion, without immediate loss of viability, subsequently cells detachment and finally death with apoptosis characteristics in MCF-7 and HeLa cell lines. These results support the finding of Avila
et al. [
14] and Bond
et al. [
15] who demonstrated a dose-dependent toxic action of PMA on pancreatic cancer cell lines. These authors also suggested that the growth inhibitory of PMA is associated with an increase in apoptosis which contributes to its anti tumor effects.
2.4. Analysis of Apoptosis by Flow Cytometry
The results of flow cytometry analysis are presented in
Table 2. These results showed that cell viability of MCF-7 and HeLa cell lines with initial values of 98.1 and 98.7% decreased significantly (
p < 0.05) to 29.7 and 31.5% upon treatment with PEs and to 26.4 and 29.5% upon treatment with PMA, respectively. The MCF-7 and HeLa cell lines showed 30.3 and 25.9% apoptotic cells upon treatment with PEs, while cells treated with PMA showed significantly (
p < 0.05) higher values at 35.4% for MCF-7 cell line. Although, the PEs appeared to be less active as compared to the PMA in induction of apoptosis, the percentage of dead cells indicated no significant difference between the cells treated with PEs and PMA. The difference in the potential of PMA and PEs in induction of apoptosis could probably due to the numbers or the position of functional groups present in the PEs structures. In addition, the comparison of apoptotic cell values in both cell lines indicated that the MCF-7 cells showed the apoptosis symptoms earlier than HeLa cells in the presence of PEs. The flow cytometry result also confirmed that PEs isolated from Jatropha meal and also PMA induced apoptosis cell death upon 24 h exposure.
Table 2.
Percentage of viable, apoptotic and dead cells analysed by flow cytometry.
Table 2.
Percentage of viable, apoptotic and dead cells analysed by flow cytometry.
| MCF-7 Cells (%) | HeLa Cells (%) | S.E.M |
---|
| Untreated | PEs-treated | PMA-treated | Untreated | PEs-treated | PMA-treated | |
---|
Viable | 98.1 a | 29.7 b | 26.4 c | 98.7 a | 31.5 b | 29.5 b | 2.78 |
Apoptotic | 1.5 d | 30.3 b | 35.4 a | 2.3 d | 25.9 c | 28.8 bc | 2.28 |
Dead | 2.2 d | 52.4 ab | 55.1 a | 2.6 d | 46.8 c | 48.9 bc | 3.47 |
2.5. DNA Fragmentation Assay
DNA fragmentation is a natural phenomenon that takes place in cells undergoing apoptosis. As shown in
Figure 6 the isolated PEs and PMA induced nucleosome-sized DNA fragmentation. The presence of DNA cleavage bands in cells treated with PEs indicated the similar cytotoxic effect of PEs to that of PMA. This result was in agreement with Day
et al. [
16] who observed changes in morphological features, apoptosis and endonuclease digestion of genomic DNA after 24 h incubation in human prostate adenocarcinoma cells (LNCaP) treated with PMA.
Figure 6.
DNA fragmentation induced by isolated PEs and PMA in MCF-7 and HeLa cancer cell lines at IC50 concentration. The extracted DNA was run on 2% agarose gel and the image was documented using Bio-Rad Gel documentation system. Lane 1: 1 kb DNA ladder; Lane 2: MCF-7+PEs; Lane 3: MCF-7+PMA; Lane 4: HeLa+PEs; Lane 5: HeLa+PMA.
Figure 6.
DNA fragmentation induced by isolated PEs and PMA in MCF-7 and HeLa cancer cell lines at IC50 concentration. The extracted DNA was run on 2% agarose gel and the image was documented using Bio-Rad Gel documentation system. Lane 1: 1 kb DNA ladder; Lane 2: MCF-7+PEs; Lane 3: MCF-7+PMA; Lane 4: HeLa+PEs; Lane 5: HeLa+PMA.
2.6. Gene Expression Analysis
The expression analyses of proto-oncogenes including c-Myc, c-Fos, and c-Jun in MCF-7 and HeLa cells upon treatment with isolated PEs and PMA are shown in
Table 3. The expression of c-Myc gene in MCF-7 and HeLa cell lines showed significant down-regulation with the value of −2.6 and −2.3 fold changes upon treatment with PEs and −3.2 and −3.6 fold changes upon treatment with PMA, respectively. Similarly, the expression of c-Jun gene in MCF-7 and HeLa cell lines was significantly down-regulated with the value of −1.3 and −1.7 fold changes after treatment with PEs and −1.7 and −2.2 fold changes upon treatment with PMA, respectively. The c-Fos gene in both cell lines was also significantly down-regulated with the value of −2.1 and −2.5 fold changes while treated with PEs and −3.2 and −3.5 fold changes after treatment with PMA, respectively.
Table 3.
Fold-changes in the expression levels of c-Myc, c-Jun and c-Fos genes in MCF-7 and HeLa cell lines upon treatment with PEs and PMA.
Table 3.
Fold-changes in the expression levels of c-Myc, c-Jun and c-Fos genes in MCF-7 and HeLa cell lines upon treatment with PEs and PMA.
Down-regulated genes | MCF-7 Cells | HeLa Cells |
---|
PEs | p 1 | PMA | p | PEs | p | PMA | p |
---|
c-Myc | −2.6 | 0.02 | −3.2 | 0.03 | −2.3 | 0.03 | −3.6 | 0.04 |
c-Jun | −1.3 | 0.04 | −1.7 | 0.02 | −1.7 | 0.04 | −2.2 | 0.03 |
c-Fos | −2.1 | 0.03 | −3.2 | 0.04 | −2.5 | 0.02 | −3.5 | 0.05 |
These genes are known as proto-oncogenes and their expression levels in the cancer cells are abnormally higher than normal cells. The proto-oncogenes are often involved in signal transduction pathway. In fact, the c-Myc gene is responsible for cell growth and proliferation, differentiation and apoptosis, while c-Fos/c-Jun complexes interact with AP-1 site on the promoter to regulate the expression of various genes involved in everything from proliferation and differentiation to defence against invasion and cell damage. The down-regulation of proto-oncogenes in this study may be mediated through the PKC family since Hatton
et al. [
17] has shown the activation of PKC is the earliest response of the cells to the presence of PMA and this activation affected the expression of downstream genes including proto-oncogenes. In line with the result of this study, Udou
et al. [
18] has also reported the role of PMA in activation of PKC which resulted in down-regulation of c-Jun gene in glandular epithelial cells.
2.7. Western Blot Assay
As shown in
Figure 7 and
Figure 8, PKC-δ protein was significantly (
p < 0.01) over-expressed in both cell lines treated with isolated PEs and PMA. The results also indicated significant (
p < 0.01) over-expression and cleavage of Caspase-3 protein in both cell lines as one of the feature of apoptosis. In fact, PEs are known as activator of PKC and their binding to PKC is the first step in activation of PKC. This binding is saturable and occurs through specific interactions within the C1 domain in the regulatory region of the PKC molecule [
11], However, the response of the cell could vary depending on the types of activated PKC. In most systems, PKC-α, ε and ι act as anti-apoptotic kinases, whereas PKC-θ, μ and δ act as pro-apoptotic kinases [
19]. In line with this result, several researchers reported the pro-apoptotic effect of PMA in different cell lines [
20,
21]. The over-expressed PKC-δ in this study confirmed the pro-apoptotic effects of PEs upon 24 h incubation, concomitant to the results of flow cytometry.
Figure 7.
Expression of PKC-δ and Caspase-3 proteins in treated and un-treated MCF-7 cell line. Cells were treated with isolated PEs from Jatropha meal and PMA at the IC50 concentration incubated for 24 h. Equal amounts of total cellular protein of treated and un-treated cells were subjected to Western blot analyses for PKC-δ, Caspase-3 and GAPDH protein expression. All values represent mean ± standard error from three independent experiments, *** p ≤ 0.001 and ** p ≤ 0.01 indicate significant difference compared to the untreated control.
Figure 7.
Expression of PKC-δ and Caspase-3 proteins in treated and un-treated MCF-7 cell line. Cells were treated with isolated PEs from Jatropha meal and PMA at the IC50 concentration incubated for 24 h. Equal amounts of total cellular protein of treated and un-treated cells were subjected to Western blot analyses for PKC-δ, Caspase-3 and GAPDH protein expression. All values represent mean ± standard error from three independent experiments, *** p ≤ 0.001 and ** p ≤ 0.01 indicate significant difference compared to the untreated control.
Caspases comprise a family of different cysteine proteases that are synthesized as inactive zymogens and are activated by proteolysis [
22]. The activation of Caspase-3 upon different apoptotic stimuli is dependant on various initiator pathways. Basically, the generation of pro-apoptotic signals in death receptors and even mitochondria could also activate an initiator of upstream caspase, which usually possesses a long NH
2-terminal prodomain such as found in caspases-8, -9 and -10. These initiator caspases can activate the Caspase-3 and results in apoptotic execution [
23]. Laouar
et al. [
24] have also reported that the activation of PKC in the presence of PMA led to activation of caspase cascade proteins and finally apoptosis in human myeloid HL-60 leukemia cells. Consequently, the apoptosis observed in this study could be the result of PKC-δ activation by PEs and PMA which resulted in down-regulation of proto-oncogenes including c-Myc, c-Fos and c-Jun genes. Down-regulation of these genes could be the reasons of activation of Caspase-3 and apoptosis execution.
Figure 8.
Expression of PKC-δ and Caspase-3 proteins in treated and un-treated HeLa cell line. Cells were treated with isolated PEs from Jatropha meal and PMA at the IC50 concentration incubated for 24 h. Equal amounts of total cellular protein of treated and un-treated cells were subjected to Western blot analyses for PKC-δ, Caspase-3 and GAPDH protein expression. All values represent mean ± standard error from three independent experiments, *** p ≤ 0.001 and ** p ≤ 0.01indicate significant difference compared to the untreated control.
Figure 8.
Expression of PKC-δ and Caspase-3 proteins in treated and un-treated HeLa cell line. Cells were treated with isolated PEs from Jatropha meal and PMA at the IC50 concentration incubated for 24 h. Equal amounts of total cellular protein of treated and un-treated cells were subjected to Western blot analyses for PKC-δ, Caspase-3 and GAPDH protein expression. All values represent mean ± standard error from three independent experiments, *** p ≤ 0.001 and ** p ≤ 0.01indicate significant difference compared to the untreated control.
Phorbol 12-myristate 13-acetate as an activator of PKC isozymes may promote tumor formation [
9] or apoptosis [
25]. Day
et al. [
16] suggested that activation of a PMA-inducible kinase(s) mediates apoptosis of androgen-sensitive prostate cells by means of an intracellular pathway that may involve the transient activation of the early response transcription factors NGFI-A and c-Fos, whereas, Fujii
et al. [
26] reported that PMA induced apoptosis in prostate cancer cells through over-expression of PKC-δ. In contrast, Park [
27] concluded that the PMA is not only a tumor promoter, but can also induce apoptosis in gastric cancer cells through activation of PKC and the activation of serine protease(s) and Caspase-3/CPP32. Indeed, the multiplicity effects of PEs on biological systems are associated with the type of PEs, type of cell, time of exposure and other experimental conditions which can affect either pro-apoptotic or anti-apoptotic activities.