Selective Cytotoxicity of Kaempferia parviflora Extracts in Human Cell Lines

With the current cancer adjuvant therapy, undesirable side effects remain a concern as drugs do not specifically target cancerous cells. Many researchers have explored various kinds of targeted therapy. It has been reported that herbs can possibly be an option for target-specific drugs. One of the most interesting Thai traditional plants is Kaempferia parviflora (KP), categorized in the Zingiberaceae family, which is commonly known as black ginger or “Krachaidum”. KP extract was considered safe with no alterations in human blood and urine chemistry parameters and has been used as a dietary supplement in traditional medicine for a long time (Wattanathorn et al., 2012; Promthep et al., 2015; Saokaew et al., 2017; Yoshino et al., 2018; Chivapat et al., 2018). Pharmacotoxic and histopathological effects were not reported in rats (Songpol et al., 2010). No genotoxicity is found in bacteria (Yoshino et al., 2019). Daily ingestion of KP extract is believed to safely reduce body fat composition, particularly abdominal fat, in Japanese overweight and pre-obese subjects. Dietary supplementation with KP Abstract


Introduction
With the current cancer adjuvant therapy, undesirable side effects remain a concern as drugs do not specifically target cancerous cells. Many researchers have explored various kinds of targeted therapy. It has been reported that herbs can possibly be an option for target-specific drugs. One of the most interesting Thai traditional plants is Kaempferia parviflora (KP), categorized in the Zingiberaceae family, which is commonly known as black ginger or "Krachaidum". KP extract was considered safe with no alterations in human blood and urine chemistry parameters and has been used as a dietary supplement in traditional medicine for a long time (Wattanathorn et al., 2012;Promthep et al., 2015;Saokaew et al., 2017;Yoshino et al., 2018;Chivapat et al., 2018). Pharmacotoxic and histopathological effects were not reported in rats (Songpol et al., 2010). No genotoxicity is found in bacteria (Yoshino et al., 2019). Daily ingestion of KP extract is believed to safely reduce body fat composition, particularly abdominal fat, in Japanese overweight and pre-obese subjects. Dietary supplementation with KP extract has been reported to inhibit body weight rising, body fat accumulation, and glucose intolerance in both obese mice fed with high fat diet (Yoshino et al., 2014) and obese type II diabetic mice (Akase et al., 2011;Shimada et al., 2011;Matsuda et al., 2014).
Many pharmacological activities were previously mentioned including anti-proliferative effects in various cancer types, i.e. ovarian (Paramee et al., 2018), cervical (Potikanond et al., 2017), gastric , leukemic (Banjerdpongchai et al., 2008;Banjerdpongchai et al., 2009), bile duct (Leardkamolkarn et al., 2009), lung (Patanasethanont et al., 2007) and melanoma (Ninomiya et al., 2016) cancer cell lines. Furthermore, KP extract can also inhibit P-glycoprotein-mediated multidrug resistance (Patanasethanont et al., 2007a;Patanasethanont et al., 2007b). These imply a promising opportunity of this herbal extract to be a supplements or potential agent in cancer adjuvant therapy. Methoxyflavones in KP have been claimed for physiological and pharmacological activities (Chen et al., 2018). A quality control assessment of KP raw materials and products was performed by a gas chromatography using 11 flavonoids as a reference (Sutthanut et al., 2007). Three main effective methoxyflavones, 3,5,7,3',4'-pentamethoxyflavone ( P M F ) , 5 , 7 -d i m e t h o x y f l a v o n e ( D M F ) , a n d 5,7,4'-trimethoxyflavone (TMF) are always mentioned for pharmacological effects (Chen et al., 2018). However, effects of KP extract in cancer cell lines with different levels of aggressiveness have never been reported yet.
This study is the first to compare the safety and cytotoxic effects of two KP extracts, aqueous and alcoholic extracts, in human cancer and normal cell lines. The extract with better cytotoxic effects would be selected for further cytotoxicity experiment in cancer cell lines varying in aggressiveness.

KP extracts
The black ginger rhizomes were purchased from Ratchaburi province, Thailand in December, 2017. KP rhizomes were identified by Dr. Wandee Gritsanapun, who retired from Department of Pharmacognocy, Faculty of Pharmacy, Mahidol University, Thailand. A voucher specimen (SWU-PY-1801) was kept at Department of Physiology, Faculty of Medicine, Srinakharinwirot University, Thailand. Processed with good manufacturing practices (GMP), rhizomes were assured by quality control of raw material. Dried KP rhizomes were mechanically powdered using electrical stainless steel blender, extracted with water thrice at room temperature, filtered and passed through spray drying process. For alcoholic extract, powdered rhizomes were extracted at room temperature with 95 % ethanol thrice, filtered and concentrated as much as possible by a rotary evaporator. The percentage yield of aqueous and ethanol extract was expressed per 100 grams of dried rhizomes as 2.5 and 5 %, respectively. Both extracts were kept in amber bottles in the refrigerator.

Chemicals and reagents
All chemicals for cell culture were purchased from Gibco-BRL (NY, USA). All other chemicals used in this study were in analytical grade and purchased from Sigma-Aldrich Co (MO, USA).

Thin layer chromatography (TLC)
Phytochemical screening was performed by TLC technique. Ready-made TLC silica gel 60F254 from Merck (MA, USA), 5 cm x 5 cm in size, as the stationary phase. Ethyl acetate was adopted as mobile phase. Quercetin and gallic acid were run in parallel with the extracts as phenolic and flavonoid standard compounds, respectively. After spotting two KP extracts and two reference compounds, the spotted TLC plate was then placed in a developing chamber containing ethyl acetate until the solvent front nearly reached the top of the plate. TLC plate was removed from the developing chamber and air-dried. Colored spots were visualized immediately under a hand-held ultraviolet (UV) lamp emitting short-wave (254 nm) and long wave (365 nm) UV light. Natural product (NP) solution was applied and visualized under 365-nm UV light.

Cell lines and culture conditions
Human urinary bladder cancer cell line (T24), three human prostate cancer cell lines, PC3, DU145, and LNCaP, as well as a non-cancer cell line, human umbilical vein endothelial cell (HUVEC), were purchased from American Type Culture Collection (ATCC). All were maintained in recommended culture media supplemented with 10% heat-inactivated fetal bovine serum, 100 units/ mL penicillin, and 100 μg/mL streptomycin, at 37°C in a humidified atmosphere of 5% CO2 and 95% air until 80-90% confluence. Cells are routinely sub-cultured twice a week.

Cell viability assay
To evaluate the safety and cytotoxicity of KP extracts in various cell lines, proliferation assay was performed using 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide (MTT) as described previously (Yahayo et al., 2013). Briefly, each type of cells was sub-cultured in 24-well plates at a seeding density of 6 x 10 4 cells per well with full culture media until 80-90% confluence. Subsequently, cells were treated with serial concentrations of KP extract in a humidified atmosphere of 5% CO 2 at 37°C for 1, 4 and 7 days. At the end of the incubation period, the cells were washed twice with phosphate-buffered saline followed by addition of and 150 μl of culture medium containing 1.0 mg/ml of MTT was added into each well and further 3-4 h incubation. The media containing MTT was removed and the blue MTT-formazan product was extracted with acidified isopropyl alcohol. Absorbance of blue crystals was quantified spectrophotometrically at 570 nm using ELISA microplate reader (Biotex-synergy-HT). Percentage of survival was plotted against control (untreated) group. Each sample was assayed in triplicate.

Quantitative real-time polymerase chain reaction (qPCR)
After treatment with ethanol KP extract, cells were harvested and total RNA was isolated using TRIzol reagent (Thermo Fisher Scientific, Inc., USA) according to the manufacturer's instruction. Complementary DNA (cDNA) was synthesized from 1.0 μg of total RNA using a SuperScript TM Reverse Transcriptase kit (Invitrogen, USA). P53 and SIRT1 gene expression was quantified by quantitative real-time PCR using CFX96 Touch Real-Time PCR Detection System (BIO-RAD, USA). Each reaction was repeated independent triplets.

Statistical analysis
All data are expressed as the mean of three independent experiments ± standard deviation compared to the control group. Least squares linear regression analysis This study firstly the first to view TLC plates under the UV light box (Camag UV Cabinet 4, USA). TLC plates can exhibit green fluorescence under short wave, 254 nm, UV light. Absorbing short-wave UV, UV-active compounds will interfere fluorescence exhibition of the plates and appear as dark bands illustrated in Figure 1. This is called a mask effect. Ethanol KP extract completely expressed more bands than aqueous KP extract. It may be due to more organic compounds extracted by ethanol. Polyphenolics, such as gallic acid, and flavonoids, such as quercetin, might be found in both extracts in different concentrations.
Two KP crude extracts were separated by traditional TLC. Plates were visualized under UV light at 254 and 365 nm the results are shown in Figure 1. The existence of polyphenolic and flavonoid compounds in different concentrations can be seen in TLC chromatogram. These are going to be the fingerprint profiles for confirming the contents in each extraction. It can be very useful for was done using Microsoft Excel to determine the IC 50 values. Comparison between groups was performed by one-way analysis of variance (ANOVA) using SPSS IBM Singapore Pte Ltd (Registration No.1975-01566-C) following by the Student's t-test. Statistical significance is determined by p-value less than 0.05.

Phytochemical study
Organic compounds found in herbal extract mostly appear color, colorless or pale on the white TLC plate which are very difficult to localize the positions. These compounds can be visualized by either non-destructive or destructive process. Allowing compounds to remain in an unchanged form, non-destructive method can be easily done under UV light. To identify the existence of proper organic compounds, staining with proper reagents allow specific color to develop.

Comparison of cytotoxicity between aqueous and ethanol extracts
Cytotoxicity of aqueous and ethanol KP extract in human urinary bladder cancer cell line (T24) was determined by MTT assay after receiving treatment for 1, 4 and 7 days. Linear segment of the dose-response curve (% survival and KP extract concentration) of three independent sample preparations was used to determine average IC 50 values. Both aqueous and ethanol extract graphs show a dose-and time-dependent manner as illustrated in Figure 2. IC 50 values of aqueous extract are 553.13 ± 20.53, 307.06 ± 7.89, and 106.06 ± 6.66 μg/mL after 1, 4 and 7 days, respectively. In contrast, ethanol extract has a lower IC 50 of 29.62 ± 1.13, 16.91 ± 0.72, and 7.56 ± 1.45 μg/mL after 1, 4 and 7 days, respectively (p<0.05).

Cytotoxicity in different aggressive human prostate cancer cells
Cytotoxicity of ethanol KP extract in three prostate cancer cell lines varying in aggressiveness is illustrated in Figure 4. Expressing a low level of androgen receptors, DU145 and PC3 are known to be poorly-differentiated androgen-independent. However, LNCaP, with a high level of androgen receptors, is classified as well-differentiated androgen-dependent.
Due to its lowest aggressiveness, IC 50 value of DU145 is the least among three prostate cancer cell lines. For LNCaP, the hormone-dependent pathway is believed to involve its cytotoxicity.

Cytotoxic activity induced by p53 and SIRT1 gene expression
Normally inactivated in cells by its negative regulators, tumor-related protein p53, encoded by a tumor suppressor gene, frequently mutates during cancer development. In Figure 5, p53 gene expression was significantly up-regulated in T24 and down-regulated in HUVEC. In contrast, Figure 6 revealed that SIRT1 gene expression was significantly down-regulated in T24 and up-regulated in HUVEC.

Discussion
Phytochemical study indicated the difference in compositions between aqueous and ethanol extract. Brief fingerprint profiles from TLC will be implemented as a reference for raw-material quality control and an explanation for different physiological and pharmacological effects found in future studies.
Dose-and time-dependent manners of T24 response are shown in Figure 2. The obviously low IC 50 value of ethanol KP extract after receiving treatment for 1, 4 and 7 days indicates a higher potency. In general, low IC 50 value   implies that a compound possesses high cytotoxicity and potency. Thus therapeutic dose for that compound will be low if implemented in clinical settings. For all of the following experiments, the authors solely used ethanol extract.
Regarding selective activity to cancerous cells, Figure 3 shows that T24 has approximately 3-7 times lower IC 50 value than that of HUVEC. This evidence possibly suggests a selective effect of ethanol KP extract to bladder cancer cell line. The gap difference of IC 50 value between HUVEC and T24 may not be sufficient to confirm the safety of ethanol KP extract in endothelial cells. Considering its low oral bioavailability of 1 to 4% reported in rats, toxic dose of KP extract in endothelial cells can be very difficult to identify (Mekjaruskul et al., 2012). However, current pharmaceutical studies have been trying to improve the dissolution rate, drug permeability and bioavailability of methoxyflavones in KP (Mekjaruskul et al., 2013;Tuntiyasawasdikul et al., 2014;Chairuk et al., 2020).
In addition, ethanol KP extract also exerts anti-proliferative activity in three prostate cancer cell lines with different metastatic potential. Lower IC 50 value was detected in lowly metastatic cells, DU145, while higher IC 50 value was found in highly metastatic cells, PC3. Supported by Paramee et al., 2018, high IC 50 value was reported in human ovarian cancer cell line, SKOV3, which is known as a high-grade serous adenocarcinoma with a high metastatic rate. The reason for high cytotoxicity in LNCaP may involve a hormone-dependent pathway. This is consistent with the previous KP extract report mentioning suppression of benign prostate hyperplasia by potent inhibition of 5α-reductase (Murata et al., 2013).
Cellular mechanism of KP extract remains unknown. General cellular mechanism was screened using gene expression analysis of p53 and SIRT1. P53, classified as a tumor suppressor gene, plays a crucial role in stabilizing other genes by preventing DNA mutations. Previously, KP extract has been reported to inhibit the expression of cell-cycle inhibitors including p53 in normal cells and human dermal fibroblasts (Park et al., 2017). Inhibition of p53 expression might possibly involve in cell survival. Moreover, KP extract also induces SIRT1 expression, a gene involving several important biological controls, which facilitates mitochondrial biogenesis to prolong cellular life span (Bai et al., 2018;Vargas-Ortiz et al., 2019). This might be the cause of higher IC 50 value in HUVEC. Figure 7 summarizes proposed mechanisms of ethanol KP extract in both T24 and HUVEC. For T24 cell line, ethanol KP extract up-regulated p53 and down-regulated SIRT1 gene. As a result, IC 50 value decreases and selective anti-proliferative activity is introduced. Apoptosis induction was also found in other cancerous cells including human ovarian cancer SKOV3 cell line (Paramee et al., 2018), human leukemic U937 cell line (Banjerdpongchai et al., 2009), and human bile duct cancer RMCCA-1 cell line (Leardkamolkarn et al., 2009).
In conclusion, compared to the aqueous version, ethanol KP extract exhibits higher anti-proliferative effect, hypothesized to be via p53 and SIRT1 gene expression, specifically to cancerous cells which is approximately 5-7 times more than normal or endothelial cells. Moreover, low bioavailability of methoxyflavones suggests its safety for consumers who may select pharmaceutical or supplementary products containing KP extracts.