Pharmacognosy Magazine

ORIGINAL ARTICLE
Year
: 2022  |  Volume : 18  |  Issue : 79  |  Page : 532--539

Anticancer potential of Piper retrofractum on breast cancer MCF-7 cells via induction of cell death and reduction of cell migration


Benjaporn Buranrat 
 Faculty of Medicine, Mahasarakham University, Muang, Maha Sarakham, 44000, Thailand

Correspondence Address:
Benjaporn Buranrat
Faculty of Medicine, Mahasarakham University, Muang, Mahasarakham
Thailand

Abstract

Background: Piper retrofractum (PR) is traditional medicine in the South-East country. Objectives: This study examined the PR effects on anticancer and anti-migratory activity in human breast cancer cells. Materials and Methods: Determination of cancer cell death by the sulforhodamine B, colony formation, flow cytometry methods. Mechanism-induced cells death was assessed by intracellular reactive oxygen species generation, caspase 3 activity, mitochondrial function, gene expression, and protein expression. Results: The ethanolic extract of PR had higher effects against MCF-7 cells than DW extract expressing the high levels of piperine. The extracts stimulated cell death in MCF-7 cells and induced apoptosis with increasing caspase 3 activity, ROS generation, and decreasing mitochondria function. Moreover, the extract also inhibited gene-related cell proliferation, cdk2, cdk4, and ckd6, and altered protein-related cell growth, cyclin D1 reduction and p21 induction. Finally, the extract caused inhibition of cells migration by reducing MMP 9 levels. The extracts of PR stimulated MCF-7 cells death in association with increasing apoptosis and inhibiting cancer cells migration. The induction of cancer cell death may be through modulating mitochondrial function. Conclusion: These extracts may be a novel strategy to improve the efficacy of chemotherapy to treat MCF-7.



How to cite this article:
Buranrat B. Anticancer potential of Piper retrofractum on breast cancer MCF-7 cells via induction of cell death and reduction of cell migration.Phcog Mag 2022;18:532-539


How to cite this URL:
Buranrat B. Anticancer potential of Piper retrofractum on breast cancer MCF-7 cells via induction of cell death and reduction of cell migration. Phcog Mag [serial online] 2022 [cited 2022 Sep 26 ];18:532-539
Available from: http://www.phcog.com/text.asp?2022/18/79/532/356399


Full Text



SUMMARY

PR extract found potential anti-cancer and anti-migratory activity against MCF-7 cells human breast cancer.

[INLINE:1]

Abbreviations used: AMC: Ac-DEVD-7-amino-4-methylcoumarin; AO/EB: acridine orange/ethidium bromide; CDK: Cell cycle dependent kinase; DMEM: Dulbecco's modified eagle medium; PI: Propidium idodide; PR: Piper retrofractum; ROS: Reactive oxygen species; SRB: Sulforhodamine B.

 Introduction



Presently, herbal remedies or plant medicine have become popular for treating several diseases, due in part to the lower risk of side effects. For several decades, anticancer drug discovery has focused on natural compounds or derivatives.[1],[2] Piper retrofractum (PR) is one of the most interested in the anticancer effect from the Piperaceae family. PR has been used as herbal medicine in Asia and South-East Asia including Thailand,[3] for long time. Several parts of PR are reported for treating/preventing many diseases such as menstrual pain, inflammation, tuberculosis, depression, respiratory tract infections, pain in the Gastrointestinal tract, arthritic conditions, hepatotoxicity, and especially cancer.[4],[5]

The ripe fruit of PR used traditionally in herbal remedies. It has been indicated that the active compounds extracted from PR are piperine, piperlongumine, piperlonguminine, pipermonaline, piperundecalidine, diaeudesmin sylvatin, and sesamin.[4] Most abundant in ripe fruit is piperine, the alkaloid which contributes to their pungency. For cancer studies, PR and piperine have been reported to inhibit cancer cells proliferation such as cervical, prostate, colon ovarian, lung, and breast cancer.[6] Moreover, they inhibit cancer cells proliferation and apoptosis through arresting at G0/G1 phase in the cell cycle with down-regulating cell cycle-dependent kinase (CDK) inhibitors, p21 and p27 expression as well as up-regulating caspase 3 enzyme level and increasing the cleavage of PARP.[7] In osteosarcoma and melanoma, piperine inhibiting at the G2/M phase and G1 phase with inhibiting cyclin B1 levels.[8] Many evidences suggested that PR or piperine caused induction the several cancer cells' death.

For more information, PR extract and piperine caused induction breast cancer cells apoptosis by unbalancing of reactive oxygen species (ROS) homeostasis,[9] as well as melanoma.[10] Furthermore, piperine induces rectal adenocarcinoma and oral squamous carcinoma cell death via modulating the mitochondrial pathway.[11],[12] The induction of caspases cascade by piperine is detected after the release of cytochrome c from mitochondria to cytosol, then activating caspase 3 and 9, the cleavage of PARP and inactivating of MAPKs pathways such as JNK and p38/MAPK.[13] On the other hand, piperine has been reported to inhibit cancer cells migration by down-regulating of matrix metalloproteinase 9 (MMP 9) expression.[14] However, the PR extract of ripe fruit on the human breast cancer cells is still less information.

Based on the previous studies, we hypothesized that the crude extract from the rip fruit of PR showed anticancer effects by reducing cell growth, increasing apoptosis, and inhibiting metastasis. In this present study, the experiment used the human breast cancer cells MCF-7 to examine whether both PR extracts by DW and EtOH against breast cancer MCF-7 cell proliferation, activate cell apoptosis, and inhibit migration. The measurement of piperine in both parts of PR extract is explored.

 Materials and Methods



Reagents

Sulforhodamine B, RIPA lysis, crystal violets were obtained from Sigma Chemical (St. Louis, MO). Antibodies for primary against cyclin D1, p21, beta-actin, and secondary were purchased from Cell Signaling Technology (Dancer, MA, and USA). Reagents for cell culture were from Gibco-BRL Life Technologies (Grand Island, New York).

Plant extraction

The ripe fruit of PR was collected from Chiang Mai Province, Thailand, in 2019 and identified by Pornpimon Wongsuwan, Assistant Professor, Faculty of Medicine, Mahasarakham University. The herbarium specimen number of this material has been deposited in the Faculty of Sciences, Mahasarakham University (No. MSUT_7235). The PR extract was extracted by distilled water (DW) and ethanol (EtOH) and made as our previous work described.[15] Briefly, dried ripe fruits (250 g) were boiled with water or macerated with 95% ethanol (7 days), filtered, evaporated, and lyophilized to obtain the dry extract. The % yield was 9.24% and 5.26% for DW and EtOH extract and the extracts were kept at -20°C until use.

HPLC method

To examine the piperine contents of both PR extracts by using the HPLC method as described previously.[15] Briefly, PR extract was loaded into C18 analytical column by using the mobile phase composed of acetonitrile: DW: Acetic acid (60.0:39.5:0.5), the injection was performed using a 20 μL loop at 340 nm.

Cell culture and cell proliferation method

MCF-7 cells, human breast cancer cell line, was cultured in Dulbecco's modified eagle medium (DMEM) added with sodium bicarbonate (NaHCO3), penicillin (100 U/mL), streptomycin (100 μg/mL), and fetal bovine serum (10%) and incubated under 5% CO2 in air at 37°C. Cancer cells were subcultured every 2-3 days after confluent using trypsin-EDTA (0.25%).

The effects of PR extract on the viability of the MCF-7 cells was measured by the SRB method.[15] Cells were plated on culture plates (1 × 104 cells/well) for 24 hr and then cells were exposed to various concentrations of PR extract both of DW and EtOH (0-500 μg/mL) for 0-72 hr. Afterward, the cancer cells were fixed, stained with 0.4% SRB for 30 min. The cells were solubilized with 10 mM Tris base solution. After that, the optical density (O.D.) was examined by spectrophotometer (540 nm).

Colony formation method

The effects of PR extracts on colony formation were measured by the colony formation method.[15] The cells were plated about 500 cells into 6-well plates overnight and the next day cells were added to the new DMEM medium containing various concentrations of PR extracts (0-250 μ g/mL) for 24 hr. Next, cancer cells were exposed to the new DMEM medium and grown for 14 days at 37°C and 5% CO2. Afterward, the cells were then fixed, stained with 0.5% crystal violet, and counted the colony formation.

Wound healing method

The effects of PR extract on the cell migration were measured by the wound healing method.[15] The cells were plated on a 24-well culture plate (2.5 × 105 cells/well) overnight and the next day cells were scratched to make a wound by using a pipette tip (0.2 mL). Afterwards, the cells were washed with PBS buffer to remove any detached cells, the wound was photographed as a baseline for 0 hr. The cells were exposed to various doses of PR extract and a series of images of the scratched wound were taken 48 hr after treatment. The distance of the wound was calculated by dividing the area by the length of the scratch.

Gelatin zymography method

The effects of PR extract on the MMP 9 expression in the culture medium were measured by the gelatin zymography method.[15] The cells were plated on a 24-well culture plate (2.5 × 105 cells/well) overnight, next day cancer cells were exposed to PR extract for 48 hr, collected the DMEM medium, centrifuged, and measured the protein concentration. The conditioned DMEM medium (20 μg) was mixed with a 2x non-reducing sample buffer and then subjected to electrophoresis on 10% polyacrylamide gels containing gelatin (1 mg/ml). Afterward, the gel was washed third time with 2.5% Triton X-100 and then exposed to developing buffer overnight at 37°C. After that, the gel was stained with 0.25% Coomassie Blue R-250 and de-stained in the de-staining buffer. The band was detected as clear bands against the blue background of Coomassie BlueR-250 and the band intensity was determined.

Acridine orange/ethidium bromide (AO/EB) method

The effects of PR extract on apoptosis were measured by AO/EB method.[16] The cells were plated on a 96-well culture plate (1 × 104 cells/well) for 24 hr, then cells were exposed to PR extract for 24 hr, washed, added to the AO/EB solution (1 μg/mL each) for 15 min at room temperature. Thereafter, cells apoptosis was determined and captured by inverted microscopy (10x magnification).

Apoptotic method by flow cytometry method

The effects of PR extract on apoptosis were confirmed by the flow cytometry method.[17] The cells were plated on a 6-well culture plate (2.5 × 105 cells/well) for 24 hr, then cancer cells were exposed to PR extract for 24 hr, washed with cold PBS buffer for three times, stained with 5 μL propidium iodide (PI) and 5 μL Annexin V FITC for 15 min on ice, and then resuspended in assay buffer, as described by the manufacturer. Cells were analyzed using a BD Accuri™ C6 Plus flow cytometer with BD Accuri™ C6 Plus software FCS 3.1 (Becton Dickinson, San Jose, CA).

Caspase 3 activity method

The effects of PR extract on caspase 3 activity were examined by the kits.[15] Cells were plated on a 6-well culture plate (2.5 × 105 cells/well) overnight, next day cells were exposed to PR extract for 24 hr, collected the cells pellets, lysed by RIPA buffer, and measured the protein concentration. Measurement of caspase 3 activity was performed by using a substrate, Ac-DEVD-7-amino-4-methylcoumarin (AMC), and AMC as a standard. The fluorescent signals were set to 360 (excitation wavelengths) and 460 nm (emission wavelengths), respectively.

Reactive oxygen species formation method

The effects of PR extract on ROS formation were measured by a DHE-fluorescent probe.[15] The cells were plated on a white 96-well culture plate (1 × 104 cells/well) for 24 hr, the cells were exposed to PR extract plus DHE probe for 90 min, and then measured the fluorescent signal using a fluorescent plate reader and the wavelengths were set to 518 (excitation) and 605 (emission) nm, respectively.

Mitochondrial membrane potential method

The effects of PR extract on mitochondrial functions were examined by JC-1 fluorescent probe.[18] Cells were plated on a white 96-well culture plate (1 × 104 cells/well) for 24 hr, the cells were exposed to PR extract plus DHE probe for 90 min, and then measured the fluorescent signal using a fluorescent plate reader and the wavelengths were set to 485 (excitation) and 535 (emission) nm, respectively.

Reverse transcription-polymerase chain reaction (RT-PCR) method

The effects of PR extract on gene expression were examined by RT-PCR.[18] Cells were plated on a white 6-well culture plate (2.5 × 105 cells/well) for 24 hr, the cells were exposed to PR extract for 24 hr and then extracted the total RNA using trizol® solution (Invitrogen, Grand Island, NY). The total RNA (1 μg) was reversed to single-stranded cDNA using iScript reverse transcriptase (Bio-Rad, Hercules, CA). The primers for PCR were shown as in [Table 1].{Table 1}

The RT-PCR was carried out using SYBR Green PCR Master Mix (Bio-Rad Laboratories, Inc., CA, USA), and thermal cycling was performed using CFX96 Touch real-time PCR detection system (Bio-Rad Laboratories, Inc., CA, USA) under the following conditions: denaturation at 95°C for 3 min and amplification by cycling 40 times at 95°C for 15 s and 60°C for 31 s.

Western blotting method

The effects of PR extract on protein expression were measured by Western blotting.[18] The cells were plated on a 6-well culture plate (2.5 × 105 cells/well) for 24 hr, the cells were exposed to PR extract for 24 hr, lysed the cancer cells, and then measured the protein concentration. The protein (20 μg) was subjected to a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Thereafter, the protein was transferred to polyvinylidene fluoride (PVDF) membrane, blocked with 2.5% albumin, and exposed to primary antibodies against cyclin D1 (1:1000), p21 (1:1000), and β-actin (1:2500) overnight. After that, the membrane was exposed to a secondary antibody for 2 hr (1:5000) at room temperature. The specific protein bands were subsequently examined by Clarity™ Western ECL Substrate (Bio-Rad Laboratories, Inc., MA, and USA).

Statistical analysis

The data were analyzed and compared between PR extract and control groups by using the Prism 5 program (GraphPad Software, San Diego, CA, USA) and expressed as mean ± SEM.

Results were considered to be statistically significant at a value of P < 0.05.

 Results



Piperin concentration in PR extract

Piperine is an active compound in the ripe fruit of PR and it has been reported that piperine caused the induction of cancer cells death and apoptosis. The two extractions of PR from DW and EtOH extract showed the higher piperine concentration in EtOH extract than DW extract [Figure 1]a,[Figure 1]b,[Figure 1]c approximately 17.02 ± 3.56 μg/mL and 1.55 ± 0.56 μg/mL, respectively. For the further experiment, this work needs to find the potency between DW and EtOH extract on MCF-7 cells death and cell migration.{Figure 1}

PR effects on cell viability

To explore the cytotoxic effect of PR extract on MCF-7 cells by using SRB and colony formation method. After cells were treated with two PR extractions for 0-72 hr and viability of all cultured cells showed a dose- and time-response manner [Figure 2]a,[Figure 2]b. MCF-7 cells were more sensitive to EtOH extract than DW extract, with IC50 values for EtOH of 177.87 ± 56.07 μg/mL, 83.81 ± 15.63 μg/mL, 67.40 ± 2.97 μg/mL and IC50 values for DW of 83.46 ± 3.31 μg/mL, 29.72 ± 72 μg/mL, 14. 55 ± 1.03 μg/mL for 24, 48, and 72 hr, respectively.{Figure 2}

The effects of PR extract on MCF-7 cells replication was determined by using colony formation. The colony formation of MCF-7 cells was inhibited by both PR extract and showed the sensitivity with EtOH extract than DW extract with IC50 values of 49.84 ± 4.51 μg/mL and 97.08 ± 16.34 μg/mL, respectively [Figure 2]c,[Figure 2]d. This suggests that PR extract has the effect to stimulate breast cancer cells death and repress the cancer cells' regrowth by dose-response manner. Moreover, EtOH extract has a higher potency than DW extract with correlating the piperine levels in the extract.

PR effects on cell apoptosis

To explore an apoptotic effect of PR extracts on MCF-7 cells and then AO/EB staining and flow cytometry methods were used. From the results obtained that both PR extracts caused the induction of cells apoptosis. EtOH extract caused a significant reduction of viable cell counts more than DW extract and accelerated apoptosis at the dose of 250-500 μg/mL [Figure 3]a. Furthermore, the data from flow cytometry showed that two PR extractions induced cancer cells to early apoptotic cells with higher than untreated control groups at a dose of 250 μg/mL [Figure 3]b.{Figure 3}

EtOH showed a higher activity than DW extract. Moreover, EtOH of PR extract caused significant induction of the caspase 3 activity at the dose of 250 μg/mL [Figure 4]a, stimulation of ROS formation at the dose of 500-1000 μg/mL [Figure 4]b, and reduction of mitochondrial membrane potential at the dose of 500 μg/mL [Figure 4]c. These data indicated that EtOH extract had a greater activity than DW extract.{Figure 4}

PR effects on gene- and protein-related cell proliferation

The effects of PR extract on gene and protein expression of cell proliferation were further evaluated. The three genes-related cell proliferation including cdk2, cdk4, cdk6, were examined by RT-PCR. The data indicated that both PR extracts significantly inhibited cdk2, cdk4, cdk6 gene expression levels [Figure 5]a. Moreover, PR extracts inhibited the protein-related cell growth, cyclin D1, stimulated protein-inhibited cell cycle, p21 protein [Figure 5]b, and finally, PR extract caused inhibited MCF-7 cell growth.{Figure 5}

PR effects on cell migration

The PR extract on MCF-7 cells migration was evaluated by wound healing assay. Results showed that the dose of 250 μg/mL of DW extract significantly suppressed MCF-7 cell migration [Figure 6]a. Similarly, exposure to EtOH extract also significantly inhibited the migration at the lower dose than DW extract, 50-500 μg/mL [Figure 6]a. Additionally, MMP 9 expression correlated with cancer cell migration and then these data indicated that both PR extracts significantly suppressed the MMP 9 levels in the DMEM culture medium after incubated with the extract for 48 hr [Figure 6]b. Both PR extracts had the greater activity to inhibit MCF-7 migration.{Figure 6}

 Discussion



Human breast cancer is cancer with rapid growth and significant metastatic potential.[19] The development of a novel effective agent targeting antiproliferation and antimetastasis could be valuable to improve current treatments in breast cancer patients.[19] These data revealed that both PR extracts had the levels of piperine, the active compound of PR extract that caused induction of many cancer cells types such as Dalton's lymphoma ascites, Ehrlich ascites carcinoma, breast, prostate, colon, and lung cancer cells.[6],[20],[21] less information on PR extract on human cancer; however, many reports have been indicated that the active compounds from PR such as piperine and piperlongumine suppressed the many cancer cells growth.[21],[22] Especially, piperine found the higher levels in Piper species; Furthermore, it induced several cancer cells death by arresting the cell cycle at the G1 phase and relating with the cdk inhibitor-1 (p21/WAF1) activation and cyclin D1 protein suppression.[10] In the same situation, the piperine interrupts the ROS homeostasis that caused induction of the damage of DNA, lipids and proteins,[10] and then leads to cancer cells death. The other active compound is piperlongumine, it has been reported that piperlongumine inhibits prostate cancer cells' growth and is associated with the reduction of cdk such as cdk2, cdk4, cyclin D1, and cyclin E, which are related to G1 phase accumulation.[23] Like this work, PR extracts found the inhibition of breast cancer cells growth with decreasing cdk2, cdk4, cdk6 gene expression and then reducing cyclin D1 and increasing p21 as well. Finally, the PR extract and its active compound were stimulated MCF-7 cell's death by several mechanisms.

Consistency with the effects of PR extracts on cdk gene expression levels; furthermore, both of the extracts had the high potency to inhibit cyclin D1 and stimulate p21 protein levels. Sriwiriyajan et al.,[24] reported that PR extract induced breast cancer cells such as MDA-MB-231, MDA-MB-468, MCF-7, MCF-12A, with very low IC50 values with 17.10 ± 0.46, 12.27 ± 2.14, 19.69 ± 0.88, 32.41 ± 5.94 μg/mL for 72 hr, respectively. The step of apoptosis is typically accomplished via two major pathways: intrinsic and extrinsic pathways in mitochondrial function.[25] In these same circumstances, it was detected that the alkaloid from PR extracts disturb the homeostasis of ROS levels that, in order, cause DNA damages,[10] with stimulated G1 phase arrest through activating the checkpoint kinase-1. On the other hand, some data found that the MDA-MB468 and MDA-MB-231, were exposed to piperine, G1 phase arrest was occurred through inactivating the G1 phase by modulating cyclin D3, cdk4, growth factors (E2F-1). The data indicated that piperine or PR extracts with higher piperine concentration may be suppressed the cancer cells proliferation and supported the chemopreventive action.

In addition, PR extracts inhibited the breast cancer cell migration with wound healing study by suppressing MMP 9 expression levels in the culture medium as well. Therefore, the extract may be a promising agent for treating human breast cancer by targeting antigrowth- and metastasis-associated events. Piper species such as Piper nigrum has been reported that inhibited breast cancer cells migration with very low concentration. The last step of cancer cells migration was triggered by a proteolytic enzymes such as MMPs, a family of Ca2+-, and Zn2+-dependent endopeptidases.[26] Piperine positively decreased the expression level and/or stimulation of MMPs and also indirectly hampers tumor spreading. As well, piperine suppressed lung metastasis of murine breast cancer model (4T1) by reduction of expression levels of MMP 9 and MMP 13.[9] Subsequently, migration was inhibited by piperine and/or PR extract and all these molecular mechanisms down-regulated MMP 9 expression.

 Conclusion



This finding revealed that both PR extracts elicit an anti-growth and anti-metastatic effect against breast cancer MCF-7 cells. Down-regulating caspase 3, inducing ROS generation, and interfering mitochondrial function contributes in part to its effect as well as reducing proliferating-gene (cdk2, cdk4, cdk6), proliferating-protein (cyclin D1), and increasing proliferating-protein inhibitor (p21) protein levels. Thus, this compound may be a potential candidate for treating metastatic breast cancer by inhibiting MMP 9 expression. Further study is required to obtain more pharmacological data and to verify the effects of in vivo study.

Highlight

This study showed that PR extract significantly inhibits human breast cancer cells proliferation, induces cancer cells apoptosis and suppresses cells migration with a low dose of PR extract.The mechanism of PR extract generated ROS formation and decreased mitochondrial function in intracellular MCF-7 cells.These results suggested that PR is a potential therapeutic agent against breast cancer.

Author's contributions

Benjaporn Buranrat designed, conducted the experiments, analyzed data, and wrote the manuscript.

Financial support and sponsorship

This work was supported by the Thai Traditional Medical Knowledge Fund (Grant No. 13/2560) and the Faculty of Medicine, Mahasarakham University (Grant No. 2560).

Conflicts of interest

There are no conflicts of interest.

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