Table of Contents    
ORIGINAL ARTICLE
Year : 2020  |  Volume : 11  |  Issue : 2  |  Page : 118-127  

Multimodality of human epidermal growth factor receptor-2 antagonism restores the apoptotic capacity of liver cancer cells


1 Department of Chemistry, Biotechnology/Biomolecular Chemistry Program, Faculty of Science, Cairo University, Giza; Department of Biotechnology, Basic and Applied Sciences Institute, Egypt Japan University of Science and Technology, Borg El-Arab, Alexandria, Egypt
2 Department of Chemistry, Biotechnology/Biomolecular Chemistry Program, Faculty of Science, Cairo University, Giza, Egypt
3 Department of Biotechnology, Basic and Applied Sciences Institute, Egypt Japan University of Science and Technology, Borg El-Arab, Alexandria; Department of Biochemistry, Faculty of Science, Ain Shams University, Cairo, Egypt

Date of Submission25-Sep-2019
Date of Decision26-Sep-2019
Date of Acceptance26-Dec-2019
Date of Web Publication22-Jul-2020

Correspondence Address:
Ahmed Osman
Department of Biotechnology, Basic and Applied Sciences Institute, EgyptJapan University of Science and Technology, Borg El-Arab, Alexandria, 21934
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jnsbm.JNSBM_183_19

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   Abstract 


Background: Hepatocellular carcinoma, the most widespread form of liver cancer and one of the most common and lethal malignancies, is characterized by poor prognosis, late onset, and a lack of clear-cut diagnostic markers. Novel therapeutic approaches are desperately required to develop effective treatment regimens. Methods: In this study, we attempted to reverse the proliferative capacity of liver cancer cells through employing a 3 – prong approach. We evaluated the antitumorigenic effects of some medicinal plant extracts that contain bioactive phytochemicals. In addition, we used Imatinib – a tyrosine kinase inhibitor (TKI), with human epidermal growth factor receptor (Her2)-specific small interfering RNA(siRNA) species to counteract the Her2-induced proliferative capacity of cancer cells. In our model, we evaluated the extent of activation of apoptotic mechanisms versus the proliferative and antiapoptotic strategies mounted by cancer cells. Results: Our results showed that HepG2 cells treated with 0.5 mM Imatinib exhibited marked downregulation of Her2 expression, upregulation of the proapoptotic marker, BAX and a downregulation of proliferative markers GPC3 and transforming growth factor (TGF)-β (45, 29, 95, and 115 folds, respectively). In the meantime, there was also significant downregulation of Her2, TGF-β, Mcl1, Spp1, GLUL and GPC3 expression and activation of apoptotic system in the cells treated with a mixture of anti-Her2 siRNA, Imatinib along with some selected extracts where the mixture successfully decreased viability of cancer cells. Conclusion: our study reveals the potential of using TKI along with phytochemical therapy and RNA interference as adjuvant regimen for treatment of liver cancer to augment the efficacy of the current control programs, yet, minimizing the side effects by transition to targeted rather than mass therapies.

Keywords: Hepatocellular carcinoma, human epidermal growth factor receptor 2, imatinib, small interfering RNA


How to cite this article:
Mousa NO, Gado M, Osman A. Multimodality of human epidermal growth factor receptor-2 antagonism restores the apoptotic capacity of liver cancer cells. J Nat Sc Biol Med 2020;11:118-27

How to cite this URL:
Mousa NO, Gado M, Osman A. Multimodality of human epidermal growth factor receptor-2 antagonism restores the apoptotic capacity of liver cancer cells. J Nat Sc Biol Med [serial online] 2020 [cited 2020 Oct 30];11:118-27. Available from: http://www.jnsbm.org/text.asp?2020/11/2/118/290482




   Introduction Top


Hepatocellular carcinoma (HCC), the frequent form of liver cancer, is the fifth most common type of cancer in the world and is considered as the fourth leading cause of cancer-related death.[1],[2] HCC commonly occurs in Southeast Asia and Africa, and also, its frequency has almost doubled in Western countries in the last 20 years[3] as it is associated with hepatitis C viral infections and alcohol-induced liver cirrhosis.[4] HCC is considered the most aggressive type of malignancies and is often diagnosed at a terminal stage, usually with survival rates of <1 year. The etiology of HCC is generally an acute hepatic insult, which progresses over time with advanced liver fibrosis and cirrhosis as typical precursors.[5]

Currently, three strategies are currently available for treatment of HCC: orthotopic liver transplantation, surgical resection, and local destruction. Nonetheless, not many patients are eligible for these curative therapies as they mainly depend on the size and site of the tumor and the extent of damage. Despite the presence of these options, the recurrence rate is relatively high.[6],[7] Thus, there is a crucial need for new effective and well-tolerated remedies to ameliorate survival among advanced-HCC patients and to achieve a longer lasting remission after the therapeutic treatments (adjuvant therapy).[8],[9]

Recent molecular studies have revealed that herbal remedies have pleiotropic effect like anti-inflammatory,[10] antiviral,[11] and anticancer effects.[12] The anticancer effects of some medicinal extracts are achieved mainly by the presence of bioactive compounds, for example, polyphenols that attenuate liver injury and act as antifibrotic and antiproliferative agents. Some herbal extracts have been studied for their remedial properties such as curcumin,[13] resveratrol,[14] and silibinin,[15] which have been proven to improve the pathological consequences of.[16]

Another treatment option for the HCC is targeted chemotherapy;[17] it is not routinely used in the treatment of HCC for reasons including the multidrug-resistant 1 gene, which undermines the efficacy of the systemic chemotherapy.[18] However, some trials are performed to make effective drugs for treating HCC.[19] For this purpose, the molecular origin of the HCC must be taken into consideration. The pathogenesis of HCC occurs through two principal mechanisms; the first is the fibrotic tissue damage that leads to cirrhosis and the second includes oncogenes activation and tumor suppressor genes mutations.[20] The two mechanisms are related to aberrations in cell signaling pathways that lead to extensive tumor vascularization.[21]

Regarding cell signaling, tyrosine kinases are important members that are involved in the signaling cascades, and hence, they have major roles in many biological processes such as differentiation,[22] metabolism,[23] and apoptosis;[24] any deregulation in their activity will contribute to the pathogenesis of human cancers. Many tyrosine kinase receptors, such as those for vascular endothelial growth factor, platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and insulin growth factor are involved in cell proliferation.[25] Human epidermal growth factor receptor (HER) 2 (17q12) belongs to the Her2 family.[26] The frequency of HER2 amplification was previously reported in patients with different types of cancer.[27],[28],[29] Several retrospective studies have reported that HER2 amplification is considered a poor prognostic marker that is associated with increased risk of invasion, metastasis, and worse survival.[30] However, there are no enough studies investigated the role of Her2 in the pathogenesis of HCC.

Imatinib (STI571; Gleevec or Glivec), a tyrosine kinase inhibitors (TKIs), acts as a semicompetitive inhibitor and exerts its inhibitory effect of tyrosine kinase enzymes by binding the enzyme near ATP binding site of such proteins which leads to locking the kinase domain in a nonfunctional form. Imatinib was found to exert similar effects in cancers with upregulated TK receptors comparable to that achieved in chronic myeloid leukemia (CML) patients.[31]

RNA interference is an evolutionary conserved system that regulate gene expression in insects, nematodes, plants, and mammalian cells via forming 21–23 bp small interfering RNA (siRNA).[32],[33],[34] Gene silencing using particular mRNA-specific siRNA molecules is poised to have an important impact on the treatment of human disorders, particularly cancers and is currently being studied in clinical trials as a potential remedy for a range of inherited diseases.[32],[33],[34],[35] siRNA is introduced into cells by using liposomes transfection in a wide range of cells, where they bind to the target mRNA and activate an RNA degradation process that leads to 80%–90% downregulation in the corresponding protein levels. Wu et al. have studied the effects of targeting BMP-2 using BMP-2 specific siRNA molecules in SMMC7721 liver cancer cells. They found that migration and invasion abilities decrease after treatment.[36],[37]

In the present study, we investigated the anti-cancer effect of some herbal extracts rich in poly phenolic, phytochemical compounds, such as quercetin, kaempferol, rutin, ellagic acid, naringenin, myricetin, and syringic acid, on HCC cells using HepG2 cell line as a model. We also synthesized Her2-specific siRNA molecules to enhance the sensitivity of liver cancer cells to imatinib as a targeted therapy.


   Materials and Methods Top


Cell culture conditions

HepG2 cell line was obtained from the American Type Culture Collection (USA). The HepG2 cells were maintained as attached monolayer culture in complete media composed of RPMI 1640 medium (Biowest, France) supplemented with 10% heat-inactivated fetal bovine serum (Biowest, France) and 10.000 U. of penicillin and streptomycin and amphotercein B (Biowest, France). The cell line was maintained in T-25 or T-75 tissue culture flasks (Greiner Bio-One, Kremsmünster, Austria) and was incubated at 37°C in 90% humidified atmosphere, 5% CO2(BINDER, USA).

Preparation of therapeutic molecules

Preparation of herbal extracts

Ethanolic extraction of saffron stigma, Panax ginseng and ginger were performed following previous protocols.[38],[39],[40] Extracts were evaporated then dissolved in ethanol/dimethyl sulfoxide (DMSO) (1:1) (Sigma-Aldrich, USA) to yield a concentration of 50 mg/ml; all extracts were sterile filtered through 0.22 μm filters prior to treatment of cell culture.

Herbal extracts' High-Performance Liquid Chromatography

Agilent1260 infinity high-performance liquid chromatography (HPLC) Series (Agilent, USA), equipped with Quaternary pump, aKinetex® 5 μm EVO C18 100 mm × 4.6 mm, (Phenomenex, USA), operated at 30°C. The separation is achieved using a ternary linear elution gradient with (A) HPLC grade water 0.2% H3 Po4(v/v), (B) methanol and (C) acetonitrile. The injected volume was 20 μL (Detection; WJD detector set at 284 nm).

Imatinib: Tyrosine kinase inhibitor

Imatinib was obtained from the local market (Novartis, Basel, Switzerland). The drug (400 mg pill) was ground to fine powder and the active drug was dissolved in filtered DMSO (DMSO, Sigma-Aldrich, USA) to prepare a stock solution of concentration of 20 mM of Imatinib and then was stored at −20°C until use. For the experimental study, serial working dilutions with different concentrations of Imatinib (0.5 mM, 0.25 mM, 0.1 mM, 0.05 mM and 0.025 mM) were prepared.

Small interfering RNA synthesis

T7-tailed Her2 primers (T7 sequence is underlined) were designed along with a T7-tailed primer pair that is amplifying a partial sequence of smad2 gene from the blood fluke Schistosoma mansoni to be used as irrelevant negative control inin vitro assays [Table 1]. Primers were ordered from Eurofins (Luxembourg).
Table 1: List of T7-tailed primers used for synthesis of small interfering RNA molecules

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cDNA samples prepared from total RNA extracted from HepG2 and S. mansoni adult worm, respectively, were used as templates in polymerase chain reaction (PCR) reactions along with the T7 tailed primer pairs using GoTaq DNA polymerase (Promega, USA) at 60°C annealing temperature. Successful amplification of expected products was confirmed by separation onto 1.5% agarose gel and purified using Illustra GFX™ PCR and gel band purification kit (GE Healthcare, USA). About 1 μg of each purified double-stranded DNA were used as a template inin vitro transcription reaction using Takara T7 RNA polymerase (Takara, Japan) following the recommended manufacturer's instructions. Double-stranded RNA products were analyzed by 1.5% agarose gel. siRNA was produced by digestion of dsRNA with ShortCut® RNase III (New England Biolabs, USA), which produce a mixture of 18–25 bp products that were used in all transfection assays using Lipofectamine 3000 (Invitrogen, USA).

Evaluation of the therapeutic effects of the different treatments

Morphological changes

Changes such as plate detachment, shrinking of the cells, and formation of apoptotic bodies were assessed to determine the extent of autophagic cell death using an inverted microscope (Olympus, Japan). Cell viability and cell count were determined through staining with trypan blue.

Cytotoxicity assay: MTT

MTT; 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide assay was performed to determine cell viability as a function of redox potential after exposure to the different treatments. HepG2 cells were cultured in 96-well plates at a cell density of 5 × 104 cells/well, incubated overnight and next day different combinations of samples were added to each well in the respective concentration of each sample. Plates were incubated overnight, and next day, MTT assay was performed as described previously[41] (Serva, Germany). Sample absorbance at 595 nm was measured using microplate reader (BioTek, USA). Cell viability was calculated using the formula: cell proliferation % = (average absorbance of treated cells/average absorbance of control cells) × 100. For all assays, control group was HepG2 cells cultured in complete media with the solvent used in sample preparation.[42]

Cytotoxicity assay: Caspase 3/9 activation assay

For caspase-3 and-9 activation assay, we followed the same method used in MTT assay, except that HepG2 cells were cultured in 24-well plates followed by treatment for 24 h with different sample combinations. Activity of caspase-3 and -9 in different cell treatments was determined using the colorimetric assay kit following the manufacturer's instructions (Promokine, Germany). Clear supernatant of cell lysates was allowed to react with the substrate peptide (DEVDpNA; Asp-Glu-Val-Asp p-nitroanilide) for 2 h at 37°C followed by taking sample absorbance against cell lysis buffer at 400 nm using microplate reader (BioTek, USA). Optical densities of different reactions were normalized to total protein concentration by bicinchoninic acid assay.[43]

Transcriptomic analysis

Extraction of RNA: Reverse transcription reaction

Total RNA was extracted from the control and treated cells using Trizol reagents (life technologies, USA) following the recommended manufacturer's instructions.[44] Five μl GeneElute reagent (Sigma Aldrich-USA) were added as an inert carrier in the RNA precipitation step to visualize the RNA pellet. RNA was dissolved in nuclease-free water, quantified spectrophotometrically and 1 μg was used as template in the reverse transcription (RT) reaction. First strand cDNA reactions were executed using 1 μg of each total RNA as a template with 500 nmoles of each oligo dT and random decamer in 20 μl reaction volume using M-MuLV reverse transcriptase enzyme (Promega Inc., USA) following manufacturer's recommendations.

Primer design

Primers for quantitative RT-PCR assays, for the 11 selected markers, were designed with considerations of the primer length between 20–24 nucleotides, annealing temperature (≈60°C), GC content (45%–60%), and the amplicon length (ranged between 100 and 180 bp). Primer pair sets were designed so that individual primers of each set are located in different exons or spanning exon-exon junctions to avoid the undesired amplification of genomic DNA byproducts. The sequences of the primers are shown in [Table 2].
Table 2: List of reverse transcription polymerase chain reaction primers used for gene expression analysis after different treatments applied

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Real-time polymerase chain reaction

Quantitative PCR reactions were performed using the specific primer pairs and GoTaq qPCR master mix (Promega Inc., USA) in a 15 μl reaction volume using Mx3000P PCR system (Agilent technologies, USA) and MxPro software version 4.1(Agilent technologies, USA) for data analysis. Data for dissociation curves were collected at the end of amplification programs to confirm reaction's specificities. Data analysis and changes in fold expression were calculated for each marker for the treated cells as compared to the untreated control cells using Livak method.[45]

Statistical analysis

One-way analysis of variance, Duncan, and Wilcoxon test were used for data analysis. All the results are expressed as the mean ± standard deviation, and P values below 0.05 were considered statistically significant.


   Results Top


Detection of the contents of herbal extracts using high-performance liquid chromatography

To detect the phenolic compounds and flavonoids in the extracts, chromatographic separation was carried out using HPLC. The detection was optimized by first using a standard mixture of phenolic compounds to ensure that the compounds were well resolved. The retention times of peaks in the HPLC chromatograph of the samples studied were compared with that of the standards to identify the unknown. The qualitative results of the different compounds recovered after methanolic extract of saffron and aqueous extract of ginger are shown in [Supplementary Table 1], [Supplementary Table 1] and [Supplementary Figure 1], [Supplementary Figure 2]. Quercetin, kaempferol, rutin, and ellagic acid were found to be the major components recovered from the aqueous extract of ginger (133, 24.3, 16.5 and 19.2 mg/L, respectively). On the other hand, naringenin, myricetin, ellagic acid, and syringic acid were the major components of the methanolic extract of saffron (137, 69.9, 117, and 45.2 mg/L, respectively).



Effect of different treatments of cell proliferation as detected using MTT assay

MTT assay was carried out in order to evaluate the anticancer effect of different treatments and to determine the optimum concentrations to be used in future experiments. Regarding herbal extracts, the cytotoxic effect of different treatments of the extracts was tested after 48 h (0.1 mg/ml, 0.5 mg/ml, 0.75 mg/ml, and 1 mg/ml). It was clear that treating the cells with such extracts significantly reduced the cell viability compared to the untreated cells [P < 0.001; [Figure 3]a. From analyzing MTT assay results for different treatments, we found that treatment of the cells with saffron reduced the viability from 100% to 41.5% using the highest concentration as a treatment; there was no difference between using 0.1 mg/ml and 0.5 mg/ml concentrations (P = 0.249). For the ginger, using 1 mg / ml resulted in significant reduction in cell viability by about 62%. However, no difference was detected when using a lower concentration (0.1 mg / ml; P = 0.99). In the meantime, both concentrations (0.5 mg/ml and 0.75 mg/ml) produced a marginal reduction (~ 30%) in cell viability. Regarding the Ginseng, viability was reduced to be 64.5%; no significant difference was detected between the untreated cells and the lowest ginseng concentration (P = 0.9), and no difference was detected between using 0.75 mg/ml and 1 mg/ml concentration (P = 0.99). Furthermore, we tested the effects of a mixture of different extracts, and we found that the saffron–ginger mixture reduce the cellular growth to 32% [Figure 3]b and [Supplementary Figure 1]. On the other hand, we tested the cytotoxic effect of Imatinib on HepG2 cellular growth, and the cell viability was reduced to 34% after treating the cells with 0.5 mM Imatinib [P < 0.001; [Figure 4] and [Supplementary Figure 2]; however, comparable effects to the untreated controls could be seen when using the low concentrations (0.025 mM).
Figure 1: High-performance liquid chromatography chromatogram of ginger containing quercetin and kampherol as major constituents

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Figure 2: High-performance liquid chromatography chromatogram of saffron containing naringenin, myricetin, ellagic acid and syringic acid as major constituents

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Figure 3: Measuring cell viability using MTT assay after treatment with herbal extracts: (a) The effect individual crude extracts; data showed that ginger and saffron significantly inhibited cell proliferation at the highest concentration (1 mg/ml). (b) The effect of different combinations of extracts on cell viability; the saffron/ginger mixtures showed the lowest cell viability among the different mixtures at concentration of 1 mg/ml of each

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Figure 4: Cell proliferation graphics determined by MTT assay after 40 h incubation with Imatinib (concentration range 0.5 mM–0.005 mM)

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Induction of caspase-3/9 activities after treatment

To test the ability of the prepared treatments on execution of apoptosis, we tested the caspase 3 and 9 activities after applying the treatments on the HepG2 cells. For the herbal extracts, the data analysis revealed that both caspase 3 and caspase 9 showed enhanced activities after the treatment with the saffron and ginger extracts, individually, and the best activity was obtained after using a mixture of both extracts; where the activities increased 5.43 times for caspase 3 (P < 0.001) and 3.98 times for caspase 9 (P < 0.001) when compared to the control group. In the meantime, ginseng extract did not affect caspase 9 levels (P = 0.537) while it unexpectedly lowered the levels of Caspase 3 (P = 0.01) [Figure 5]a and [Supplementary Figure 3] and [Supplementary Figure 4]. Imatinib, on the other hand, increased Caspase 3 and 9 activities 5.6 and 3.9 folds, respectively (P < 0.001), after using 0.5 mM Imatinib, while after using 0.25 mM, the levels increased 4.87 and 3.2, respectively (P < 0.001) [Figure 5]b. Furthermore, the lower concentrations showed insignificant changes in caspases activities than untreated control. Our third modality for targeting Her-2 was gene silencing when using Her2-siRNA. Analysis revealed significant reduction in caspase 3 activity compared to the control cells (P = 0.011); but, it was lower than the values obtained after imatinib and herbal extracts treatments. Finally, using Her2-siRNA did not affect the levels of caspase 9 (P = 0.42) [Figure 5]c.
Figure 5: Determination of caspase 3 and 9 activities following cell treatment with: (a) herbal extracts, (b) different doses of imatinib, (c) small interfering RNA. The activity of caspase-3 and-9 is expressed as the ratio of caspase-3 and -9 activation levels as compared to the control level

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Effect of different treatments on gene expression

The next step in our study was to evaluate the modulation in gene expression that was exhibited upon treatment of the cells for 48 h with different modalities. [Figure 6] shows the fold change in gene expression of some proliferative and apoptotic markers. Our data showed that treating HepG2 cells with saffron and ginger greatly reduced the expression of Mcl1, transforming growth factor (TGF)-β, SMAD4, Her2, NELF-b, Glul, GPC3, and Spp1, while such treatment greatly enhanced the expression of the proapoptotic gene Bax [values of fold change are presented in [Figure 6]a. After Imatinib treatment, there was a significant downregulation in the expression of Mcl1, TGF-β, SMAD4, Her2, NELF-b, Glul, GPC3, and Spp1 using 0.5 mM imatinib concentration (P < 0.001), while the lower imatinib concentrations (0.05) exhibited insignificant change as compared to the control cells [Figure 6]b. Furthermore, the Her2-siRNA treatment led to extreme downregulation in Her2, SMAD4 in particular (76.9 folds and 20 folds downregulation than that of untreated HepG2 cells, respectively; P < 0.001) as shown in [Figure 6]c. Using a mixture of siRNA, herbal extracts and Imatinib showed a better effect and more downregulation of the Mcl1, TGF-β, SMAD4, Her2, NELF-b, Glul, GPC3, and Spp1 [Figure 6]d. The measured expression levels of the selected genes after applying different treatments are tabulated in [Supplementary Table 3].
Figure 6: Quantitative reverse transcription polymerase chain reaction analysis of Mcl1, Bax, transforming growth factor-b, SMAD4, Her2, NELF-b, Glul, GPC3, and Spp1 gene expressions in HepG2 cells treated with different modalities. (a) Treatment of cells with ginger, saffron, and a mixture of both extracts. (b) treatment of cells with 0.5 mM and 0.05 mM of Imatinib. (c) Treatment of cells with human epidermal growth factor receptor 2 - small interfering RNA. (d) Treatment of cells with different combinations of the 3 targeting modalities (herbal extract(s) + Imatinib + human epidermal growth factor receptor 2- small interfering RNA)

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   Discussion Top


HCC is one of the most aggressive malignancies that is in most of the cases occurs as a consequence to prolonged liver damage and cirrhosis. Worldwide, HCC is considered one of the top leading causes of death,[46],[47] and this is mainly attributed to late diagnosis that would leave the patient with few or no treatment options. This, beside the fact that current therapeutic approaches show low efficacy,[48] highlights the necessity of finding new multidisciplinary therapies that would combine chemotherapeutic agents, targeted and adjuvant therapies to prevent, control and cure HCC and delay the deterioration in liver function.[49]

In this study, we attempted to assess the efficacy of a multi-modality treatment option for HCC. The Study design employed a 3-prong approach; using herbal extract(s), a TKI along with siRNA molecules, targeting Her-2 transcript, a cellular proliferative molecule.

Our hypothesis focused on targeting Her-2, which was found to exhibit a remarkable expression in liver cancer cell line, HepG2. The three-prong Her-2 targeting strategy included the use of herbal extract that showed to contain components that exhibit marked tyrosine kinase inhibition activity. In addition to the herbal extracts that could act as natural remedies, our approach included one of the first specific TKIs, Imatinib, as well as a mixture of Her2-specific siRNA molecules (Her2-siRNA). We assessed the validity of our hypothesis by evaluating cell viability and/or the efficacy of inducing apoptotic mechanism(s) via a comprehensive repertoire of assays that could verify the effect(s) of different treatment formulations.

One of the cancer hallmarks is sustaining proliferative signaling, which enables uncontrolled growth of cancer cells, is usually achieved via abnormal activation of tyrosine kinases. Thus, a surging trend in cancer management, which is gaining momentum nowadays, is the use of TKIs as targeted therapy for maintaining liver status in HCC patients as well as in various types of cancer.[50],[51],[52],[53] In this study, we have investigated the ability of Imatinib to treat HCC using HepG2 cell line as a model for the disease. Interestingly, we observed that treating the HepG2 cell line with imatinib results in decreasing cancer cell viability and increase the activity of Caspase 3 and 9, in addition to the modulation of the levels of some proliferation, signaling and apoptotic markers. Imatinib is one of the oral targeted TKIs that act through inactivating many dysregulated receptors like c-KIT, the nonreceptor tyrosine kinase ABL and PDGF receptor alpha.[54] Lately, Imatinib was used for the treatment of neoplasm of the Gastro-Intestinal tract (GIT)[55] and CML.[56] These class of therapeutic molecules are now used for treating several types of Cancer as genomic studies identified many mutations that affects tyrosine kinase receptors like HER2 and epidermal growth factor receptor.[57]

Next to TKI and Imatinib treatment, the second treatment line in our approach is to use phytochemicals from herbal extracts. Herbal extracts are part of the control mechanisms that provide protection from cancer onset and can prevent disease development. A lot of studies were conducted to explore the potential of herbs to cure or prevent cancer progression. In this study, we tested three natural extracts: saffron, ginger, and ginseng. Both saffron and ginger showed a great potential in controlling and inhibiting the growth of cancer cells. Both herbs successfully affected the viability of the HepG2 cells which was detected microscopically and using MTT assay. Furthermore, both extracts increased the caspase 3 and caspase 9 which are important apoptotic members inside the cell. Saffron and ginger significantly increase apoptotic marker BAX and decreased ARC the anti-apoptotic marker. Furthermore, saffron and ginger greatly reduce HCC cells proliferation and inhibited many signaling cascades through downregulating CTNN-β, TGF-β, SMAD-4, GLUL, GPC3, SPP1, and NELF-β.

saffron or Crocus sativus is a natural herb that have many biologically active components like safranal and crocin and many other components that can act as antioxidant, anticancer, and anti-inflammatory.[58] Saffron is also known to treat spasms and depression.[58],[59],[60] Many studies showed that saffron have antiproliferative effects and induce apoptotic response in differentin vitro models of cancer; however, further studies are needed to find the exact mechanism by which these responses are initiated.[60] In our study, HPLC analysis of methanolic extracts of saffron revealed the presence of other phenolic components beside the major ones, like naringein, myricetin, and kaempferol. Naringein was shown to inhibit the migration of lung,[61] breast,[62] and bladder.[63],[64] In one of the studies, naringein was found to sensitize HER2 positive cancer cells to cell death through inhibiting HER2 tyrosine kinase activity.[65] Furthermore, myricetin was found to have antitumorigenic effect in many types of cancer like ovarian cancer,[66] colon[67],[68] and prostate cancer.[69] A study was conducted to study myricetin effect on tyrosine kinases, and myricetin was a potent inhibitor to tyrosine kinases met and hepatocyte growth factor.[70] The previous study showed similar results on tyrosine kinases upon using Kaempferol,[70] which is involved in the cell cycle control and apoptosis in various types of Cancer.[71],[72],[73] A recent study in 2019 showed that kaempferol is a potent chemotherapeutic agent that can be used to overcome colon cancer.[74] The components of saffron confirmed our hypothesis that saffron can be used as sole or adjuvant therapy to treat HCC. This was in agreement with Liu et al., who discovered that saffron treated HCC cells had higher Bax/Bcl-2 ratio.[75]

Furthermore, we investigated the components of ginger (Zingiber officinale Roscoe). Ginger is known to contain many biologically active compounds such as gingerol and shogoal. Like saffron, many studies were conducted on testing the anti-inflammatory, antitumorigenic, and antiproliferative effects of ginger, and it showed promising results in many types of cancer.[76],[77],[78],[79],[80],[81],[82] In our study, ginger had the same effects as saffron on liver cancer cell. It decreased the viability of the cells, increased the activity of caspase 3 and 9, and also modulated the expression of the tested markers. Similar studies were obtained as it inhibited the proliferation and induce apoptosis in different types of cancer.[83],[84],[85],[86] In addition, ginger can be used as chemopreventive agent of colorectal cancer[87] and ovarian cancer.[88] The HPLC analysis of ginger showed a high content of quercetin; a flavonoid that is present in some fruits and vegetables. Quercetin is known to have antiviral, anti-inflammatory, and antioxidants effects in addition to their ability to treat cancer through preventing G2/M cell cycle progression leading to cell cycle arrest.[89],[90] They also have the potential to act as tumor preventive agent.[91],[92],[93] Interestingly, Quercetin is also know to exhibit inhibitory effect against many tyrosine kinases in many cell lines including ABL1, Aurora-A,-B,-C, CLK1, FLT3, JAK3, MET, NEK4, NEK9, PAK3, PIM1, RET, FGF-R2, PDGF-Rα and PDGF-Rß, which are involved in the control of mitotic processes.[94]

The third approach in our study is through utilizing RNA interference. One of the key players in the progression of Cancer are a family of receptors called HER; HER. The HER family are involved in many signaling pathways that organize cellular proliferation and growth as well as participate in the survival of cells.[95],[96]

The four members of this family are cell surface receptors with tyrosine kinase domain and a ligand binding domain; however, it is can be activated constitutively without ligand binding or after dimerization with HER1 or HER3. HER2 upregulation was observed in many forms of malignancies such as breast, colon, and other types.[97],[98] Furthermore, amplification of HER2 was found to be associated with liver cancer and also found in HepG2 cell line.[99]

Targeting HER2 is now used as an approach to treat different forms of cancer by blocking the activity of overexpressed HER2 like using Trastuzumab antibody that binds HER2 extracellular segment that consequently lead to the suppression of downstream signaling pathways and inhibition of tumor proliferation.[31],[100]

In our attempt to block the over-expression of HER2 activity in HCC cells, we utilized RNA interference therapeutic molecules through designing HER2-specific siRNA molecules which was delivered into the HepG2 cells through liposome-based transfection reagent. The siRNA molecules exert their action through binding with the complementary mRNA and recruitment of RNA-induced silencing complex which consequently lead to halting protein production due to the degradation of the target mRNA.[101] The analysis showed the synthesized HER2-specific siRNA did not affect the activity of the apoptotic markers, however, the transcription levels of HER2 and other proliferative markers were significantly downregulated upon treatment with the HER2-siRNA. In agreement with our results, Faltus et al. in their study which implied utilization of siRNA that targets HER2/neu in breast cancer cells showed that HER2/neu-upregulation is essential for cellular proliferation and blocking of the target mRNA lead to arrest of cellular growth at G0/G1 phase.[102]


   Conclusion Top


This study highlights the potential of using herbal therapy along with RNA interference as adjuvant regimen to augment the efficacy of the current control programs, yet, minimizing the side effects by transition to targeted rather than mass therapies. Our approach can be used as immunotherapy to prevent the growth of liver cancer cells.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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