Coptidis Rhizome inhibits cell growth in HONE-1 cell line by inducing cell cycle arrest and apoptosis


 Background Nasopharyngeal carcinoma (NPC) is among the most common head and neck malignancies seen among adults in Malaysia. Therefore, discovery of novel anti-cancer herbal drugs is of importance. In this study, the cytotoxic effect was conducted on a traditional Chinese herbal prescription (Xiao Xian Xiong Decoction (XXXD) that is made up of 3 Chinese herbal medicines, namely Huanglian (Coptidis Rhizome), Banxia (Pinellia Rhizome), Gualuo (Fructus Trichosanthis).Methods The cytotoxic effect of the individual herb and in combination of two and three herbs was studied on 8 nasopharyngeal cancer cell lines. Global gene expression analysis was carried on extracted RNA using nCounter XT Gene Expression Assay.Results TWO-1, TWO-4, HONE-1, SUNE-1, CNE-2, HK-1, CNE-1 and C666-1 treated with Huanglian, the IC50 values obtained were 24.48, 11.77, 4.48, 10.72 6.32, 11.10, 6.77 and 27.30 µg/ml, respectively. For combination of Huanglian and Banxia, the IC50 values obtained were 74.09 µg/ml (TWO-1), 25.80 µg/ml (TWO-4), 38.10 µg/ml (HONE-1), 29.46 µg/ml (SUNE-1), 19.0 µg/ml (CNE-2) and 20.12 µg/ml (HK-1) but did not exert 50% cell killing in CNE-1 and C666-1 cell lines. The IC50 value attained for the combination of Huanglian and Gualuo was 40.70 µg/ml in HONE-1 cell line. The IC50 values obtained for XXXD (triple combination of Huanglian, Banxia and Gualuo)-treated in HONE-1 and CNE-2 cell lines were 88.55 and 92.42 µg/ml, respectively. Out of all these 7 groups of herbal samples, Huanglian showed the highest cytotoxicity against 8 NPC cell lines with the lowest IC50 value of 4.48 µg/ml recorded in HONE-1. Global gene expression showed Huanglian significantly downregulated genes associated with cell cycle arrest and apoptosis, and thus inhibit HONE-1 cell growth.Conclusions This study suggest that Huanglian could be a potent anticancer herb targeting HONE-1 cancer cell line.

and olfactory dysfunction [4]. These side effects limit the use of chemotherapeutic agents despite the high e cacy in treating speci c or target malignant cells. Due to the abundance of side effects of the existing medicines, it is wise to look for more alternative medicines for better prognosis. It is believed that natural plants could be the useful sources of new anti-cancer agent [5]. Chinese herbs are now being used as an adjunct to Western medical interventions. Chinese herbal medicine is characterized by "Holistic regulation," in which the organism is considered as a whole. With the equilibrium of the human body as the guidelines, practitioners of Chinese herbal medicine pay more attention to the diseased patients rather than the suffered diseases itself. Particular western medicine always has a strong effect on a speci c disease with focusing on a speci c physiological target and neglecting the speci c characteristics of each patient [6].
In this study we investigated the cytotoxic effect of single and combination of two and three Chinese herbal medicine on NPC cancer cell lines. Xiao Xian Xiong Decoction (XXXD) is a Chinese Medicine prescription originates from "Shang Han Lun", a renowned Chinese medical masterpiece written by Master Zhang ZhongJin of ancient Chinese Han Dynasty (216A.C.). The decoction is made up of three Chinese medical herbs derived from natural plants, namely Huanglian (Coptidis Rhizome), Banxia (Pinellia Rhizome), and Gualuo (Fructus Trichosanthis). Huanglian initially recorded in the Shennong Bencao Jing (Emperor Shennong's Classical Medical Material) and listed as one of the best herbs [7]. Berberine (BBR), an isoquinoline alkaloid, is the major active component found in in Huanglian with multiple pharmacological activities including antimicrobial, antiviral, anti-in ammatory, cholesterollowering and anticancer effects [8]. Banxia in the perspective of TCM, it is acrid, warm, and toxic in nature. It is mainly used to treat coughing, dyspnea, dizziness, nausea and vomiting, fullness in chest and palpitation caused by phlegm and uid retention [9]. Gualuo is a owering plant and the fruit is used to clear body heat and transform phlegm-heat, unbind the chest, reduces abscesses and dissipate nodules [10]. This plant can be used possibly to dissolve the protein coat on the RNA of the AIDS virus [11].
As decoction XXXD has the effect of clearing away heat, resolving phlegm, relieving stu ness in the chest and dispersing stagnancy [12]. In the past centuries, it had been used to treat many diseases involving digestion, respiration and cardiovascular disorder. In recent years, the clinical application of XXXD has been widened, especially in the aspects of inhibiting tumour growth and promoting body immunity [13]. The modern pharmacological studies have shown XXXD comprises of distinctive clinical effects, such as anti-in ammation, anti-ulcer, anti-cancer and immunity enhancement [13]. Previous invitro laboratory research on tumour cell line has shown this decoction consists of inhibitory effect against Ehrlich ascites carcinoma (EAC) and lung cancer (NSCLC) [14,15].
Previous researches have shown the anti-cancer effect of each individual constituent of XXXD. Nevertheless, the therapeutic value of XXXD on NPC has never been scienti cally scrutinized. Overall, this research comprises the preparation of aqueous-extraction of individual and combination extracts of Huanglian, Banxia, and Gualuo and subsequently screening the cytotoxicity of these herbal extracts on a panel of 8 NPC cell lines, namely TWO-1, TWO-4, HONE-1, SUNE-1, CNE-2, HK-1, CNE-1 and C666-1 with 3-(4, 5-dimethylthiazol-2-gl)-2, 5-diphenyl-tetrazoliumbromide (MTT) assay. Further gene expression analysis was carried out to quantify transcript levels of 770 genes representing 13 canonical cancer pathways in Huanglian-treated HONE-1 cell line.  Table 1 shows the combination of the extracts for the extracts preparation. The amount of Huanglian, Banxia and Gualou used is based on a ratio of 1:2:5 as stated in Shan Han Lun. All the herbs were soaked in 500ml of deionized water for an hour. Then, the herbs were boiled for half hours in a 3L customized boiling device. After cooling, the supernatant was collected and ltered with whatman paper. The ltrates were lyophilized and kept in -20 o C prior use.

Material
Cultivation of NPC cancer cell lines A total of 8 NPC cancer cells were used in this study ( 2, HK-1 and C666-1 cells were cultured in RPMI-1640 medium whereas TWO-1 and CNE-1 were cultured in DMEM. All these mediums were supplemented with 10% FBS. Cells were grown as monolayers and were maintained in a humidi ed CO2 incubator at 37°C.

Determination of Optimal Cell Seeding Concentration
MTT assay was used to determine the optimum cell seeding density of each cell line. A total of 2.5x10 3 , 5x10 3 , 10x10 3 , 20x10 3 and 40x10 3 and 80x10 3 cells/ml were seeded in a 96-well plate and incubated for 72 hours prior to cytotoxicity evaluation by MTT assay. Each well was read at 570 nm for absorbance. Absorbance versus cell density graph was plotted. The optimum cell concentration was determined approximately at the middle point of exponential phase.

MTT assay
All the herbal extracts were tested for their cytotoxicity effect on a panel of NPC cell lines. A total of 100µL of cells at the optimum cell concentration were seeded into each well of 96-well plate. Three wells containing 100µL medium only were included as a negative control and three more wells containing 100µL cell solution (untreated with herbs) serve as positive control. After 24 hours of incubation, each well was added with 10µL mixture of fresh medium containing respective herbs named as L1 to L7 at the concentrations of 100, 50, 25, 12.5, 6.25 and 3.125 µg/ml via MTT assay and then topped up with 90 µL of medium. The cytotoxicity of the herbal extract was determined at 72 hours of incubation. After 72 hours, the solution was discarded and re lled with 200 µL of fresh medium. 20 µL of MTT was then added into each well and incubated for 3 hours. After 3 hours, the plate was centrifuged in 3000 rpm for 5 minutes in 5°C Then, 180 µL of the solution was removed and 200 µL of DMSO was added. The mixture was swirled gently and kept in dark for 15 minutes. The assays were conducted in triplicate and repeated in three independent experiments. The average absorbance against number of cells/mL graph was plotted and IC 50 value was calculated.

RNA extraction
Total RNA was extracted with Direct-zol™ RNA Miniprep Plus (Zymo Research, CA, USA) following the recommendations of the manufacturer. Brie y, 600μl of TRI-Reagent® was added to pellet and homogenized completely. 600μl of ethanol 95% was directly added to the solution and homogenized. The sample was added to Zymo-Spin IIC™ column with the collection tube and centrifuged at 16,000xg for 30 seconds at 4°C. The column was moved to a new collection tube and the collection tube containing the ltrate was discarded. The RNA samples were treated with DNase. Next, 400μl Direct-zol™ RNA, a prewash was added to the column, and centrifuged. Subsequently, the ltrate was discarded and this step was repeated. 400μl RNA wash buffer was added to the column and it was centrifuged.. The column was carefully transferred from the collection tube into the RNase-free tube, 50μl DEPC-treated nuclease-free water was added to it and it was centrifuged. Extracted RNA was stored frozen at <-70°C. Purity and concentration of the total RNA were determined by spectrophotometry using the 260 nm/280 nm ratio (NanoDrop 1000, Thermo Scienti c, USA) and 1% agarose gel electrophoresis.

Nanostring sample preparation
The preparation of hybridization was carried out using nCounter XT Gene Expression Assays (NanoString Technologies, Seattle, WA). Initially, 70 μL of Hybridization Buffer was added to Reporter CodeSet to create a Master Mix. Secondly, the hybridization reaction was set up by adding 5 μL of pre-prepared RNA samples to 8 μL of Master Mix to make up 13 μL of total volume in a tube. Thereafter, 2 μL of Capture ProbeSet was added to the tube before making the nal volume of 15 μL. The tube was then capped, well mixed and brie y spinned. Finally, a total of 12 tubes (triplicate at time point of 0, 4, 8, 12 hours) were incubated in thermocycler at 65°C for about 16 hours. The next steps was puri cation and immobilisation by using the nCounter MAX/FLEX system which was carried out in the nCounter Prep Station to remove excess probes and immobilize target-probe complexes onto nCounter Cartridges. The cartridges were scanned on the nCounter Digital Analyzer for 2.5 hours. Reporter Code Count (RCC) le was an output le generated by this nCounter instruments. One RCC le was produced for each sample tested; one le contains the barcode counts from each gene and control in the CodeSet. A Reporter Library File (RLF) is a le speci c to the CodeSet. It provides nCounter instruments and the nSolver 4.0 software application with valuable information about the CodeSet, such as the assignment of probe to gene.
After importing to nSolver 4.0 software (NanoString Technologies), the RCC data les will be stored under the corresponding RLF le CodeSet on the Raw Data tab. All samples were normalized using the geometric mean of the housekeeping genes RBM45-mRNA, SLC4A1AP-mRNA, AGK-mRNA, FCF1-mRNA, MRPS5-mRNA, DDX50-mRNA, HDAC3-mRNA, EIF2B4-mRNA, NUBP1-mRNA. Differentially expressed genes between the treated and the untreated control cells were given as fold change expression. Gene expression data were mapped onto KEGG pathway graphs by Path view function of the PCPAA, providing intuitive views of regulation at the pathway level. Regulated KEGG pathways were identi ed via Nanostring Advance Analysis software. The common differentially expressed genes at the different time point, in both the upregulated and the downregulated gene lists, were identi ed by using Venny online tool.

Optimal cell density
After being incubated for 72 hours, each cell line was tested for its optimal cell density. Table 3 shows the results obtained for the optimum cell seeding density of each NPC cell line. The optimum cell seeding density of each cell line is ranging from 2.5 x 10 3 to 5 x 10 3 cells.

Cytotoxicity of the herbal extract L1 to L7 via MTT assay
The values of IC 50 (µg/mL) attained for cytotoxic effects of L1, L2, L3, L4, L5. L6, L7 against 8 NPC cell lines are summarised in Table 4 and illustrated in Figure 1.   Table 5. Among upregulated gene set, genes in MAPK signalling pathway appeared 3 times in the top 10 list (Table 5) (Figure 6b). Genes involved in a number of cancer pathways were enriched among the top differentially expressed genes as shown in Table 7.
Among upregulated gene set, genes in MAPK pathway appeared 5 times in the top 10 list (Table 6), followed by the P13K (4 times), RAS and cell cycle (2 times) as well as JAK-STAT, WNT and transcriptional misregulation pathway (1 time). Among downregulated gene sets, genes in cell cycle appeared 5 times with mostly in apoptosis pathway, followed by P13K (4 times), MAPK and RAS (2 times) as well as DNA damage-repair, TGF-beta, transcriptional misregulation pathway (1 time) ( Table 7). found in between three treatment time.

Discussion
This study was designed to uncover alterations in gene expression patterns and pathway mechanisms involved in nasopharyngeal carcinoma and subsequent comparison at different time point upon treated with Huanglian.
Methods of extraction of medicinal plants include solvent extraction, distillation method, pressing and sublimation according to the extraction principle and etc. In this experiment, aqueous-extraction of Huanglian was adopted. It was a simple, economical and effective method for oral consumption and faster absorption in body for curing diseases. This study was intended to characterize the temporal dynamics of gene expression in HONE-1 when treated with Huanglian at 0, 4, 8, 12 hrs to illuminate the series of progressive biological mechanisms involved in HONE-1 proliferation. The genes expressed in MAPK signalling pathway was upregulated at 4 and 12 hours while the genes expressed in WNT signalling pathway was upregulated at 8 hours. The cell cycle was downregulated at all time point starting from 0 until 12 hours. The MAPK pathways relay, amplify and integrate signals from a diverse range of stimuli and elicit an appropriate physiological response including cellular proliferation, differentiation, development, in ammatory responses and apoptosis in mammalian cells. MAPK pathway activation has ambiguous effect on tumour cells fate depending on cell type or cancer stages [21]. Upregulation of ERK activators NGF and BDNF was observed in HONE-1 cell at 4 hours. MAPK/ERK pathway activation is a common feature of NPC and these pathways are stimulated by the EBV-encoded LMP1 and LMP2A genes to promote the migration and invasion of NPC cells [22,23]. Furthermore, high expression of phosphorylated ERK in primary NPC is related to lymph node metastasis and radioresistance [24]. These information suggest activation of MAPK/ERK from 4 to 12 hours in this study. Similarly, activation of WNT signalling pathway also correlated with nature of NPC cell lines. Upregulation of MMP7, PLCB4, LEF1 and FOSL1 genes were observed at 8 hours post treatment. WNT signaling pathway is one of the essential signaling pathways regulating cell proliferation and differentiation. It is composed of various signal molecules, ligands and receptors such as Wnt protein and β-catenin, and is very conservative in evolution [25]. Zeng et al in 2007, concluded Wnt signaling pathway may be abnormally activated in NPC due to abnormal accumulation of WNT5A and nuclear β-catenin [26].
The cell cycle is the series of events that takes place in a cell leading to its division and replication. Regulation of the cell cycle involves processes crucial to the survival of a cell, including the detection and repair of genetic damage as well as the prevention of uncontrolled cell division. One of the most important tumour suppressors is tumour protein p53, which plays a key role in the cellular response to DNA damage [27]. In this experiment, Huanglian downregulated genes involved in cell cycle pathway in HONE-1. p53 could be activated and bind with DNA that further resorted to DNA repair and triggered to cell apoptosis. Huanglian decreased the expression of HDAC1, SMC1A, RAD, AKT3, CDC7, CCNA2, CCNE1/2 and CDC6 that induces cell cycle arrest and apoptosis, and thus subsequently inhibited cell proliferation of HONE-1.

Conclusion
In a nutshell, the ndings of this study indicate that Huanglian induces dose-dependent responses in HONE-1 that involve the up-regulation of a large group of genes associated with MAPK and WNT signaling pathways and the downregulation of genes associated with cell cycle arrest and apoptosis. This reciprocal regulatory mechanism provides clues to further understand the mechanism driving growth inhibition in NPC HONE-1 cancer cells treated with the Huanglian. Availability of data and materials The datasets analyzed during the current study available from the corresponding author on reasonable request. All authors read and approved the nal manuscript.           Venn diagram depicting the common genes differentially regulated in HONE-1 cells after treatment at different time point. Downregulated and upregulated genes in HONE-1 cells after exposure to L1.