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ORIGINAL ARTICLE
Year : 2020  |  Volume : 16  |  Issue : 68  |  Page : 28-38  

Anticancer effects of piperine-free Piper nigrum extract on cholangiocarcinoma cell lines


1 Department of Biomedical Sciences, Faculty of Medicine; The Excellent Research Laboratory of Cancer Molecular Biology, Prince of Songkla University, Hat Yai, Songkhla, Thailand
2 Department of Pharmacology, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla, Thailand
3 Department of Biomedical Sciences, Faculty of Medicine, Prince of Songkla University, Hat Yai, Songkhla, Thailand

Date of Submission01-Aug-2019
Date of Decision30-Sep-2019
Date of Web Publication31-Mar-2020

Correspondence Address:
Potchanapond Graidist
Department of Biomedical Sciences, Faculty of Medicine, Prince of Songkla University, Hat Yai, Songkhla
Thailand
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/pm.pm_288_19

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   Abstract 


Background: Black pepper (Piper nigrum L.) is widely used as a traditional medicine, including usage for pain relief, fevers, as well as an anticancer agent. Previously, we reported that piperine-free P. nigrum extract (PFPE) inhibited breast cancer in vitro and in vivo. Objective: In this present study, we explored the anticancer effects of PFPE on cholangiocarcinoma (CCA). Materials and Methods: 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed to analyze cytotoxic potential of PFPE whereas deoxyribonucleic acid (DNA) fragmentation followed by Western blot analysis were used. Results: PFPE composed of alkaloid, flavonoid, amide, lignans, opioid, and steroid. This crude extract represented cytotoxic effect against CCA cells which stronger than dichloromethane P. nigrum crude extract and piperine, especially on KKU-M213 (median inhibition concentration [IC50] at 13.70 μg/ml) and TFK-1 (IC50at 15.30 μg/ml). Interestingly, PFPE showed lower cytotoxicity against normal human cholangiocyte MMNK-1 cells (IC50at 19.65 μg/ml) than KKU-M213 and TFK-1 cells. Then, the molecular mechanisms of PFPE were firstly evaluated by DNA fragmentation followed by Western blot analysis. The degradation of DNA was observed on KKU-M213 and TFK-1 cells after treatment with PFPE at day 2. Then, proliferation proteins including topoisomerase II, AKT8 virus oncogene cellular homolog, avian myelocytomatosis virus oncogene cellular homolog, cyclin D1, signal transducer and activator of transcription 3, cyclooxygenase-2, and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) were decreased and p21 was increased. Furthermore, apoptotic proteins, such as tumor protein p53, Bcl-2-associated X protein, and p53 upregulated modulator of apoptosis were upregulated. Meanwhile, antiapoptotic protein B-cell lymphoma 2 was down-regulated. Conclusion: These results indicated that PFPE inhibited CCA through the down-regulation of cell proliferation and induction of apoptosis pathway.

Keywords: Anticancer, apoptosis, cell proliferation, cholangiocarcinoma, Piper nigrum


How to cite this article:
Tedasen A, Khoka A, Madla S, Sriwiriyajan S, Graidist P. Anticancer effects of piperine-free Piper nigrum extract on cholangiocarcinoma cell lines. Phcog Mag 2020;16, Suppl S1:28-38

How to cite this URL:
Tedasen A, Khoka A, Madla S, Sriwiriyajan S, Graidist P. Anticancer effects of piperine-free Piper nigrum extract on cholangiocarcinoma cell lines. Phcog Mag [serial online] 2020 [cited 2020 Jun 1];16, Suppl S1:28-38. Available from: http://www.phcog.com/text.asp?2020/16/68/28/281693



SUMMARY

  • piperine free Piper nigrum extract (PFPE) inhibited cholangiocarcinoma (CCA) cell lines
  • PFPE induces CCA cells to undergo apoptosis and cell cycle arrest via the inhibition of topoisomerase II
  • PFPE inhibit cell growth through the inhibition of nuclear factor kappa-light-chain-enhancer of activated B cells.




Abbreviations used: PFPE: Piperine free Piper nigrum extract; CCA: Cholangiocarcinoma; DPCE: dichloromethane P. nigrum crude extract; NMU: N-nitrosomethylurea; ER: Estrogen receptor; MMP-9: Matrix metalloproteinase-9; MMP-2: Matrix metalloproteinase-2; VEGF: Vascular endothelial growth factor; GC-MS: Gas chromatograph-mass spectrometer; MTT: 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyltetrazolium bromide; DMSO: Dimethylsulfoxide; IC50: Median inhibition concentration; MCLE: Methanol crude extract of Curcuma longa; DNA: Deoxyribonucleic acid; STAT-3: Signal transducer and activator of transcription 3; COX-2: Cyclooxygenase-2; NF-kB: Nuclear factor kappa-light-chain-enhancer of activated B cells; c-Myc: Avian myelocytomatosis virus oncogene cellular homolog; Akt: AKT8 virus oncogene cellular homolog; Bcl-2: B-cell lymphoma 2; p53: Tumor protein p53; Bax: Bcl-2-associated X protein; PUMA: p53 upregulated modulator of apoptosis.


   Introduction Top


Cholangiocarcinoma (CCA) is an epithelial cancer originating from the bile ducts with features of cholangiocyte differentiation.[1] There are 2 types of CCA (based on its location) including intrahepatic and extrahepatic.[2] For over the past four decades, incidence of CCA has been increased in United States of America,[3] Australia, England,[4] and Northeastern Thailand.[5] There are several risk factors for CCA, including primary sclerosing cholangitis, liver fluke infections (Clonorchis sinensis and Opisthorchis viverrini), choledochal cysts, Caroli's disease, hepatitis B and C infection, obesity, cirrhosis and hepatolithiasis.[5],[6] The therapeutic for CCA are limited and no current effective treatment because the majority of patients present with advanced stage disease.[7] Even treatments with advances in surgical techniques, chemotherapy and radiotherapy, the 5-year survival rate of patients after diagnosis still remain about 10%.[8] Although surgical resection has improved in the survival of most patients, the recurrent disease was found within 2 years after tumor resection.[9] Chemotherapy and radiation therapy are ineffective and show various side effects such as harmful to normal cells and bone marrow suppression.[10] Therefore, effective therapeutic and alternative treatments with no serious side effect for CCA are urgently needed.

P. nigrum L. belongs to family Piperaceae and can be used as antiapoptotic, antibacterial, anticolon toxin, antidepressant, antifungal, antidiarrhoeal, antiinflammatory, antimutagenic, antimetastatic, antioxidative, antipyretic, antispasmodic, antispermatogenic, antitumor, antithyroid, ciprofloxacin potentiator, cold extremities, gastric ailments, hepatoprotective, insecticidal, intermittent fever, and larvicidal activities.[11] The chemical constituents of P. nigrum are aromatic essential oils, alkaloids, amides, prophenylphenols, lignans, terpenes, flavones, and steroids.[12] Ethanolic crude extract of P. nigrum consists of high total phenol content shows antioxidant and anti-inflammation as well as cytotoxic property against colorectal carcinoma cell lines.[13] Using ethanol and high pressure (200 bar), P. nigrum crude extracts exhibits cytotoxicity against MCF-7 with median inhibition concentration (IC50) of 14.40 ± 3.30 μg/ml and represents tumor inhibitory effect in mammary adenocarcinoma mouse.[14] Previously, we reported that piperine-free P. nigrum extract (PFPE) strongly inhibited breast cancer MCF-7 cells with IC50 value of 7.45 μg/ml. Moreover, PFPE inhibited tumor growth in N-nitrosomethylurea-induced mammary tumorigenesis rats without liver and kidney toxicity.[15] Interestingly, PFPE upregulated tumor protein p53 (p53) and downregulated estrogen receptor, E-cadherin, matrix metalloproteinase-9 (MMP-9), MMP-2, avian myelocytomatosis virus oncogene cellular homolog (c-Myc) and vascular endothelial growth factor (VEGF) in vitro and in vivo.[16] In this present research, we further explored the phytochemical component, investigated cytotoxicity and molecular mechanisms of PFPE on CCA cell lines.


   Materials and Methods Top


Preparation of piperine free Piper nigrum extract

Seeds of P. nigrum L. were collected from Songkhla province in Thailand. The plant specimen (voucher specimen number SKP 146161401) was identified by Asst. Prof. Dr. Supreeya Yuenyongsawad and deposited in the herbarium at the Southern Centre of Thai Traditional Medicine, Department of Pharmacognosy and Pharmaceutical Botany, Prince of Songkla University, Thailand. PFPE was prepared as previously described. Briefly, grounded 250 g of dried seeds of P. nigrum L. were soaked in 300 mL of dichloromethane and incubated at 35°C for 3 h in a shaking incubator. After filtration with Whatman filter paper No. 1 and concentration using rotary evaporator, the dark brown oil residue of extracts was obtained and then recrystallized with cold diethyl ether in an ice bath to get rich of yellow crystals (piperine) and obtain brown oil residue (PFPE).[15] PFPE was kept in a desiccator until used.

Phytochemical analysis and identification of bioactive constituents by gas chromatograph-mass spectrometer

The analysis of the phytochemical screening and composition of PFPE extracts were carried out using a Gas Chromatography-Agilent 7890B combination with an Agilent 5977A triple quadrupole mass spectrometer (Agilent Technologies Inc, USA). Gas chromatograph-mass spectrometer (GC-MS) analysis is a common confirmation test, which used to make an effective chemical analysis. The PFPE samples were evaluated phytochemicals such as a flavonoids, tannins, alkaloids, steroids, phenols, glycosides, lignans, and terpenoids. An inlet temperature of 280°C with the split ratio 7:1 was employed and the helium was used as the carried gas at the constant flow rate of 7 ml/min. The oven temperature was initially maintained at 60°C for 5 min and increase at a rate of 5°C/min to 315°C for 15 min. For MS detection, an electron ionization mode was used with an ionization energy of 70 eV, ion source temperature of 230°C, and scan mass range m/z 35–500. The components were identified based on a correlation of the recorded fragmentation patterns of mass spectra that provided in the GC-MS system software version Wiley10 and NIST14. All procedures were performed at Scientific Equipment Center, Prince of Songkla University, Songkhla, Thailand.

Measuring total phenolic, tannin, flavonoid content and radical scavenging activity

The total phenolic content was determined based on Folin–ciocalteu method. Gallic acid was used as the standard and total phenolics were expressed as mg gallic acid equivalent/mg extract (mg GAE/mg extract). Total condensed tannin was measured based on HCL-vanillin method and catechin was used as the standard. The total tannin was reported as mg catechin equivalent/mg extract (mg CE/mg extract). The total flavonoid content was determined by aluminum chloride solution (AlCl3) colorimetric method. Quercetin was employed as the standard and expressed the total flavonoids as mg quercetin equivalent/mg extract (mg QE/mg extract). 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) radical scavenging activity was performed according to the DPPH trolox assay and reported as mg trolox equivalent antioxidant capacity/mg extract (mg TEAC/mg extract). All procedures were performed at Center of Excellence in Natural Products Innovation, Mae Fah Luang University, Chiang Rai, Thailand.

Cell lines and culture conditions

Three CCA (KKU-100, KKU-M213 and KKU-M055) and one cholangiocyte (MMNK-1) cells were kindly donated by Dr. Mutita Junking (Faculty of Medicine, Mahidol University, Bangkok, Thailand). TFK-1 cells were obtained from RIKEN BioResource Center and HuCC-T1 cells were obtained from the Japanese Collection of Research Bioresources Cell Bank. Mouse fibroblast, L-929 cells, were kindly donated by Associate Professor Dr. Jasadee Kaewsichan (Department of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, Prince of Songkla University, Songkhla, Thailand).

KKU-100, KKU-M213, KKU-M055, MMNK-1 and L-929 cells were grown in DMEM medium (Invitrogen), which contained 10% of fetal bovine serum (Invitrogen), 2 mmol/L of L-glutamine (Invitrogen), and an antibiotic mixture of 100 units/mL of penicillin and 100 μg/mL of streptomycin (Invitrogen). TFK-1 and HuCC-T1 cells were grown in RPMI 1640 (Invitrogen) supplemented with the same supplement as for DMEM. All cells were maintained by incubating in a 5% CO2 atmosphere, at 37°C and 96% relative humidity.

In vitro cytotoxicity

The cytotoxicity assay was performed in 96-well plate. KKU-100, KKU-M055, and MMNK-1 cells were seeded at a density of 5 × 103 cells/well. KKU-M213, TFK-1, and HuCC-T1 cells were seeded at a density of 7.5 × 103 cells/well and L-929 cells were seeded at a density of 8 × 103 cells/well. After incubation for 24 h, cells were treated with PFPE at various concentration for 48 h. The cells were then washed with 1X PBS and incubated in 100 μl of 0.5 mg/ml of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution at 37°C for 30 min. Under light protection, the purple crystals of formazan or MTT metabolites were dissolved with 100 μl of dimethyl sulfoxide and incubate at 37°C for 30 min. The absorbance was measured at 570 and 650 nm using a microplate reader spectrophotometer (Spectra Max M5, Molecular Devices), and the IC50 values were calculated.[17] According to US NCI plant screening program, a crude extract is generally considered to have in vitro cytotoxic activity with IC50 value ≤20 μg/ml.[18]

Deoxyribonucleic acid fragmentation analysis

KKU-M213 and TFK-1 cells in their exponential growth phase were seeded into 6 cm culture plate at a density of 2.5 × 105 cells/plate for 24 h and then treated with PFPE at 3 folds of IC50 values. After treatment for 96 h, cells were harvested by trypsinization. Cell pellets were lysed using the extraction buffer (containing 0.7 M NaCl, 17 mM SDS, 10 mM Tris-HCl (pH 8.0) and 2 mM EDTA (pH 8.0)) and fragmented deoxyribonucleic acid (DNA) in the supernatant was extracted once with an equal volume of phenol: chloroform: isoamyl alcohol (25:24:1) and once with chloroform: isoamyl alcohol (24:1). The DNA was precipitated with a two-thirds volume of cold isopropanol followed by centrifugation at 8,000 ×g and washed once in 70% ethanol. Finally, DNA pellet was resuspended in deionized water and analyzed by 1.5% agarose gel electrophoresis.[19]

Western blot analysis

KKU-M213 and TFK-1 cells were seeded into 6 cm culture plate at a density of 2.5 × 105 cells/plate for 24 h and then treated with PFPE at IC50 values. After treatment, cells were harvested every day for 4 days. Then, cell pellets were lysed using the RIPA buffer (containing 150 mM NaCl, 50 mM Tris, pH 7.4, 1% (v/v) NP-40, 0.25% (w/v) sodium deoxycholate and 1 mM EDTA). Total protein samples (150 mg) were loaded on 12% of SDS-polyacrylamide gel electrophoresis and transferred onto a 0.45 mm nitrocellulose membrane (Bio-Rad, 162-0115). Membrane was blocked at room temperature for 1 h with 5% non-fat milk in 1X TBS-T and then washed with 1% non-fat milk in 1X TBS-T. Membrane was incubated with primary antibodies against topoisomerase II, Bcl-2-associated X protein (Bax), B-cell lymphoma 2 (Bcl-2), p53 upregulated modulator of apoptosis (PUMA), p21, AKT8 virus oncogene cellular homolog (Akt), cyclooxygenase-2 (COX-2), Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB), signal transducer and activator of transcription 3 (STAT-3), cyclin D1 and p53 proteins. The membrane was then incubated with secondary horseradish peroxidase-conjugated antibodies. Bound antibodies were developed by a chemiluminescence detection kit using the SuperSignal West Dura Extended Duration Substrate (Thermo Scientific) and detected using a Fusion FX vilber lourmat, CCD camera (Fisher Biotechnology). GAPDH was used to normalize protein loading. Protein levels were expressed as a relative ratio to GPADH.

Statistical analysis

The median inhibition concentration (IC50) data was acquired by SoftMax 1 Pro 5 program (MDS Analytical Technologies Inc., California, USA). Student's t-test was used to analyze intergroup differences. A P < 0.05 was considered to be statistically significant. All results were represented as the mean ± standard deviation (SD). The values were obtained from at least three independent experiments.


   Results Top


Total phenolic, tannin, and flavonoid contents

Phenolics, flavonoids, and tannins are one class of secondary plant metabolites which represented anticancer activity of plant. As present in [Table 1], PFPE contained phenolic, tannin and flavonoid lower than methanol crude extract of Curcuma longa (MCLE). However, the cytotoxicity of PFPE against breast cancer MCF-7 cells (IC50 value at 7.45 ± 0.6 μg/ml) not significantly lower than MCLE (IC50 value at 5.74 ± 1.48 μg/ml). Therefore, we performed GC-MS in next experiment to identify the chemical compounds in PFPE.
Table 1: Total phenolic, tannin and flavonoid contents in piperine free Piper nigrum crude extract

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Phytochemical screening

In this study, the phytochemical analysis using GC-MS was carried out. The chromatogram and predicted constituents are shown in [Figure 1] and [Table 2].
Figure 1: Gas chromatograph-mass spectrometer chromatogram of piperine free Piper nigrum extract

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Table 2: Chemical constituents in piperine free Piper nigrum extract

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Results showed that PFPE contained five chemical groups including alkaloids, terpenes, amides, lignans, opioid and steroid with 17, 13, 7, 3, 1, and 1 compounds, respectively. The highest percentage of peak area of each group were pipercitine (21.66%, alkaloid), caryophyllene (13.28%, terpene), acrivastine (2.34%, amide), kusunokinin (1.28%, lignan), methyldihydromorphine (1.18%, opioid), and beta-stigmasterol (1.74%, steroid) which showed the anticancer activity.

Effect of piperine free Piper nigrum extract on the viability of cholangiocarcinoma, cholangiocyte and normal fibroblast cell lines

The cell viability of CCA and normal cell lines was measured using the MTT assay. All cell lines were incubated with extracts for 48 h. The IC50 values represented the mean ± SD of three different experiments. Among these cell lines, PFPE showed the highest cytotoxicity against KKU-M213 cells with IC50 value of 13.70 ± 1.14 μg/ml. Moreover, PFPE demonstrated cytotoxic effect stronger than dichloromethane P. nigrum crude extract (DPCE) (IC50 at 22.22 ± 0.26 μg/ml) and piperine (IC50 at 27.01 ± 0.36 μg/ml). The positive reference drug (doxorubicin) showed a very strong cytotoxic activity on normal and almost cancer cells. Surprisingly, doxorubicin showed same cytotoxic activity with PFPE against TFK-1 cells [Table 3].
Table 3: Cytotoxicity of piperine free Piper nigrum extract against cholangiocarcinoma, cholangiocyte and normal mouse fibroblast cell lines

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Piperine free Piper nigrum extract induces deoxyribonucleic acid fragmentation on KKU-M213 and TFK-1 cells

A DNA fragmentation assay was used to determine whether the action of PFPE was associated with apoptosis or not. Apoptosis can be visualized as a ladder pattern of 180-200 base pairs due to DNA cleavage by the activation of a nuclear endonuclease enzyme. Since, PFPE demonstrated a strong cytotoxic effective on KKU-M213 and TFK-1 cells, both cell lines were used to determined DNA fragmentation. As shown in [Figure 2], the DNA ladder pattern was observed at day 2 after exposure with 3 folds of IC50 concentration of PFPE.
Figure 2: Analysis of Deoxyribonucleic acid fragmentation induced by piperine free Piper nigrum extract in KKU-M213 and TFK-1 cell lines. Cells were treated with piperine free Piper nigrum extract for 4 days and Deoxyribonucleic acid fragmentation was assessed by 1.5% agarose gel electrophoresis and ethidium bromide staining. KKU-M213 (a) and TFK-1 (b) cells were treated with 41.10 and 45.90 μg/ml of piperine free Piper nigrum extract, respectively. The data are representative of three independent experiments carried out under the same conditions

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Piperine free Piper nigrum extract inhibited proteins associated with inflammation that induces bile duct cancer

In this experiment, we determined proteins associated with inflammation that induced bile duct cancer including STAT-3, COX-2 and NF-kB using Western blot analysis. KKU-M213 cells were treated with 13.69 μg/ml of PFPE and incubated for 96 h. The results showed that the STAT-3, COX-2 and NF-kB protein levels were reduced in a time dependent manner and significantly decreased at 48-96 h [Figure 3]a and [Figure 3]c. Furthermore, TFK-1 cells were treated with 15.29 μg/ml of PFPE and incubated for 96 h cells. The STAT-3 and COX-2 protein levels were significantly reduced at 72-96 h in a time-dependent manner. The NF-kB protein was decreased significantly at 24 and 72 h [Figure 3]b and [Figure 3]d.
Figure 3: Expression of inflammation-related proteins in KKU-M213 (a and c) and TFK-1 (b and d) cells treated with piperine free Piper nigrum extract at 24, 48, 72 and 96 h. The levels of signal transducer and activator of transcription 3, cyclooxygenase-2 and Nuclear factor kappa-light-chain-enhancer of activated B cells and GAPDH proteins were measured using the Western blot analysis. Densitometric analysis normalized to GAPDH. Data were represented as mean ± standard deviation and three independent experiments were done. *P < 0.05 compared with control group (0 h)

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Piperine free Piper nigrum extract inhibited proteins involved in the cell proliferation and growth

Proteins related to cell proliferation and growth of bile duct cancer cells, including topoisomerase II, Akt, c-Myc, cyclin D1, and p21 were examined after treatment with PFPE using IC50 concentration of each cells. The result showed that topoisomerase II was significantly decreased at 24 h and p21 was increased at 96 h in KKU-M213 cells [Figure 4]a and [Figure 4]c. Meanwhile, PFPE treated TFK-1 cells showed a significant decreased in topoisomerase II at 72 h and p21 was increased at 24 h [Figure 4]b and [Figure 4]d. Then, Akt protein was decreased at 48 and 72 h in KKU-M213 and TFK-1 cells, respectively. Moreover, c-Myc and cyclin D1, a protein that worked after those proteins, were found significantly decreased at 48-96 h in both cell lines [Figure 4].
Figure 4: Effect of piperine free Piper nigrum extract on cell growth and cell cycle arrest. KKU-M213 (a and c) and TFK-1 (b and d) cells were treated with Median inhibition concentration concentration of piperine free Piper nigrum extract for 24, 48, 72 and 96 h. Then, the levels of topoisomerase II, AKT8 virus oncogene cellular homolog, avian myelocytomatosis virus oncogene cellular homolog, cyclin D1 and p21 proteins were investigated using Western blot analysis. Fold change of each protein was measured by densitometry quantitation using ImageJ software and normalized with GAPDH. P < 0.05 of three independent experiments was considered to indicate a statistically significant differences compared to control group (0 h)

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Piperine free Piper nigrum extract inhibited proteins associated with apoptosis

In this study, proteins associated with apoptosis pathway including antiapoptosis (Bcl-2) and apoptosis (p53, bax, and PUMA) were evaluated. After giving PFPE at IC50 concentration for 48 h, death cells were observed and Bcl-2 was decreased in both cells, KKU-M213 and TFK-1 [Figure 5]. In addition, the levels of p53 and Bax proteins were significantly increased at 96 h and PUMA protein was increased from 24 to 48 h in KKU-M213 cells [Figure 5]a and [Figure 5]c. Moreover, p53, Bax and PUMA were increased significantly at 24 h TFK-1 cells [Figure 5]b and [Figure 5]d.
Figure 5: Effect of piperine free Piper nigrum extract on apoptosis. KKU-M213 (a and c) and TFK-1 (b and d) cells were treated with Median inhibition concentration concentration of piperine free Piper nigrum extract for 24, 48, 72 and 96 h. Then, the levels of tumor protein p53, B-cell lymphoma 2, Bcl-2-associated X protein and PUMA proteins were investigated using Western blot analysis. Fold change of each protein was measured by densitometry quantitation using ImageJ software and normalized with GAPDH. P < 0.05 of three independent experiments was considered to indicate a statistically significant difference compared to control group (0 h)

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   Discussion Top


The incidence of bile duct cancer or CCA has increased in Thailand and chemotherapy is not sufficient to treat the aggressive type of this cancer.[5] Therefore, medicinal plants could be an alternative treatment for bile duct cancer. There are many medicinal plants that cause cell cycle arrest and apoptosis in CCA such as Tripterygium wilfordii, Atractylodes lancea (Thunb) DC., Zingiber officinale Roscoe, Phyllanthus emblica, Terminalia chebula Retz., Moringa oleifera, and Curcuma longa Linn.[20],[21]Piper species is one of medicinal plant that also shows anticancer effect, such as Piper sarmentosum,[22]Piper longum,[23]Piper chaba[24] and P. nigrum.[17] In previous study, we reported that PFPE showed anticancer activity against breast cancer in in vitro and in vivo.[15],[16] Here, we further explored the biological activity of PFPE on bile duct cancer and found that PFPE exhibited anticancer activity against CCA cell lines, especially TFK-1 and KKU-M213, a moderate differentiation with p53 mutation and well differentiation CCA cells, respectively. Using GC-MS technique, many active phytochemicals were founded in PFPE including alkaloids, terpenes, amides, lignans, opioid and steroids. Pipercitine, guineensine, and pipersintenamide, (an alkaloid compounds) represented percentage of peak area at 21.66, 10.17, and 5.65%, respectively. Pipercitine shows toxicity against larvae of Aedes aegypti,[25] and guineensine has an anticancer property against the mouse lymphoma cell line L5178Y with IC50 values of 17.0 μM.[26] Pipersintenamide, isolated from Piper sintenense Hatus, shows anticancer activity against leukamia P-388 and promyelocytic leukemia HL-60 cell lines with IC50 values of 3.78 and 3.80 μg/ml.[27],[28] Moreover, caryophyllene (13.28% in PFPE), a bicyclic natural sesquiterpene, exhibits antiproliferative effects against colorectal cancer cells (IC5019 μM) though clonogenicity, migration, invasion and spheroid formation.[29] A beta-stigmasterol (1.74% in PFPE), a steroid compound, demonstrates inhibitory effects with IC50 values of 11.14 and 18.28 μM against human myeloid leukemia K562 and prostate cancer PC3 cell lines, respectively.[30] In this recent study, we found a very potent compounds in the PFPE including piperlonguminine (4.77%), kusunokinin (1.28%), and cubebin (0.28%), which have been reported as anticancer agents.(-)-Kusunokinin and piperlonguminine, a natural lignan and alkaloid compounds, inhibited breast cancer cells (MCF-7 and MDA-MB-468) and colorectal cells (SW-620) through down-regulation of topoisomerase II and up-regulation of of p53, p21 protein levels.[31] (-)-Cubebin, a lignan compound, represents anticancer effect against myeloid leukemia, lung and nasopharyngeal cancer.[32] Interestingly, we found that PFPE showed stronger cytotoxicity against CCA cells than DPCE and piperine [Table 3]. However, piperine, the major alkaloid compound in P. nigrum, still remained in the PFPE at 5.09% [Table 2]. Similarly, CP2 (PFPE) exhibited IC50 values of 7.45 ± 1.59 μg/ml in MCF-7 cell lines, which was better than DPCE (IC50 at 23.46 ± 1.10 μg/ml).[17] These results indicate that PFPE, less piperine, was a potential crude extract in anticancer.

O. viverrini excretory/secretory products and O. viverrini antigen induce the expression of TLR4, IL-6, IL-8, TLR2, NF-κB, iNOS and COX-2 causing damage to biliary epithelium.[68] In this current study, PFPE showed down regulation of NF-kB, STAT-3 and COX-2 proteins [Figure 2]. In cancer cells, NF-kB and STAT-3 are major transcription factors that regulate proliferation, inflammatory, angiogenesis, invasive and apoptosis resistance by induction of several proteins, such as cyclin D, cyclin E1, CDK2, CDK4, CDK6, c-myc, tumor necrosis factor alpha, interleukin-1 (IL-1), IL-6, IL-8, VEGF and MMP-9.[69] NF-kB and STAT-3 proteins are induced by IL-6 to stimulate COX-2 expression in the inflammation process and cell cycle,[70],[71] which associate to CCA progression. Therefore, suppression of NF-kB, STAT-3 and COX-2 proteins cause cancer growth inhibition. Piperlongumine, an alkaloid from P. longum reduces NF-kB and c-Myc protein levels and inhibits binding of NF-kB with DNA at promoters in lymphoma cancer cells.[72] Moreover, piperlongumine also reduced the phosphorylation of JAK-1, JAK-2 and STAT-3 in gastric cancer cells.[73] Matrine, an alkaloid from Sophora flavescens Ait., significantly inhibits the viability by reduction the phosphorylation levels of JAK-2 and STAT3 proteins in CCA cells.[74] Curcumin, a natural extracted polyphenol from C. longa, also suppresses proliferation in human biliary cancer cells through inhibition of NF-kB, STAT-3 and JAK1 proteins.[75]

There are many evidences on genes and proteins which relate to bile duct cancer growth and progression, such as p53 mutation, inactivation of p21 and activation of Ras and MAPKs proteins.[76] Here, we found that PFPE could inhibit CCA cancer proliferation by decreasing of topoisomerase II, Akt, c-Myc, cyclin D1, and increasing of p21 protein levels [Figure 4]. Topoisomerase II is an enzyme involved in the DNA replication process that controls cell cycle with peaking at G2/M phase.[77] Therefore, down regulation of topoisomerase II by PFPE could induced DNA damage, interrupted cell growth and caused cell death on KKU-M213 and TFK-1 cells. Most of the clinically active agents, including etoposide (lignan) and doxorubicin (alkaloid) are topoisomerase inhibitors.[78] Previously andrographolide analogue 3A.1 from Andrographis paniculata, a diterpenoid lactone, induces cell cycle arrest by down-regulation of CDK6 and cyclin D1 in KKU-M213 cell lines.[79] Surprisingly, PFPE also exerted a significant reduction of Akt protein leading to decreasing of c-Myc and cyclin D1 and increasing of p21 levels [Figure 6]. Akt and cyclin D1 stimulate the cell cycle progression from G1/S phase to G2/M phase.[80] β-caryophyllene oxide, a terpene compound from P. nigrum, shows down-regulation of downstream of AKT pathway, including cyclin D1, COX-2 and VEGF and also up-regulation of p53 and p21 proteins in human prostate and breast cancer cells.[81]
Figure 6: The anticancer mechanism of piperine free Piper nigrum extract in cholangiocarcinoma

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In this study, we founded that the PFPE induced cell death by causing DNA fragmentation, increasing apoptotic proteins (p53, Bax and PUMA) and decreasing Bcl-2 protein levels [Figure 5]. p53, a tumor suppressor and transcription factor, is initially induced when DNA damage and takes responsibility to activate several apoptotic genes, such as Bax, PUMA and NOXA.[82],[83],[84] Similarly, ethanolic extract of P. nigrum has antiproliferative effect on MCF-7 cells, antitumor effect in vivo and triggering apoptosis via p53 and Bax and decreasing of Bcl-2 proteins.[55] Curcumin effectively induces apoptosis in CCA (CCLP-1 and SG-231) cells by stimulation of Notch1, Hes-1 and survivin apoptotic proteins.[85] Andrographolide analog 3A.1 has cytotoxicity with IC50 of 8.0 μM on KKU-M213 cells at 24 h after treatment and induces apoptosis via induction of cleaved PARP-1, Bax, caspase-3, and p53.[79] Matrine stimulates apoptosis in CCA cells through induction of cytochrome c releasing from mitochondria and reduction of caspase-3 and-9 activity.[74] Taken together, PFPE can be a potential candidate for CCA treatment in future. However, study in CCA in vivo and clinical trial need to be carried out.


   Conclusion Top


PFPE showed strong cytotoxicity against KKU-M213 and TFK-1 cell lines with IC50 values of 13.70 ± 1.14 and 15.30 ± 0.18 μg/ml, respectively. PFPE suppressed inflammation through down-regulation of NF-kB, STAT-3 and COX-2. Moreover, PFPE inhibited CCA cells growth and proliferation by down-regulation of topoisomerase II, Akt, c-Myc and cyclin D and up-regulation of p21. Furthermore, PFPE triggered apoptosis through inhibition of Bcl-2 and induction of p53, Bax and PUMA levels as summarized in the [Figure 5]. In summary, PFPE can be served as a promising crude extract for CCA treatment.

Acknowledgement

The authors thank Prince of Songkla University for the equipment funding (the Excellent Research Laboratory (ExLab004)).

Financial support and sponsorship

This work was supported by the research fund (REC 60-279-04-2) Faculty of Medicine and the Postdoctoral Fellowship, Prince of Songkla University.

Conflicts of interest

There are no conflicts of interest.



 
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