Background

Human cancer cell lines are a coherent tool to explore mechanisms associated with proliferation, deregulation, apoptosis, somatic alterations, cancer therapeutics, and pharmacogenomics. These cell lines are characterized by a broad range of mutations in cancers of different tissue origin, exhibit interrelated chromosomal losses/gains with exclusive mRNA expressions. This, in turn, allows effective extrapolation of discovered results to accurately hypothesize clinical response (Ferreira et al. 2013; Niu and Wang 2015). Cancer cell lines are generally materialized as in vitro experimental model for cancer research since these cells grow as monolayer homogeneous cell population. Other approaches such as the use of primary tumors and tumors derived from the xenografts represent mixed cell types and warrant certain manipulations to investigate DNA methylation and transcriptional profiling. Furthermore, in vivo drug testing is practically difficult and necessitates ethical clearance to execute in animals (Ferreira et al. 2013). Thus, two dimensional (2D) cell cultures are preferred in vitro models to uncover various biomolecular mechanisms that underlie cancer cells in vivo.

In 2D culture process, cell lines are usually generated through tumor tissue or by genetic transformation of primary human cells (e.g., liver) via modulating agents such as SV40, considering their proliferative potential and static metabolism. Generally, these cells are seeded on collagen treated culture plates to form confluent monolayers for development of a physiologically and pathologically specific 2D model. 2D model does not only consider cell line as selection criteria, but other elements such as, culture platform (glass or polystyrene) for mechanical assistance, growth factors requirement, utilization of essential nutrients in media, and other biophysical and/or biochemical cues also play an important role in generating a reliable disease model (Duval et al. 2017).

Traditionally 2D human hepatocarcinoma HepG2 cell cultures have been used as in vitro liver model for toxicity studies. These hepatoma epithelial cells were derived from a 15 year old Caucasian American male with well differentiated hepatocellular carcinoma (HCC) with modal chromosome number 55 and cultured in Dulbecco’s modified Eagle’s medium (ATCC 2017). Genetic profile of these cells along with activation of Wnt signaling and deregulation of cell survival pathways resemble embryonal and fetal hepatoblastoma. This cell line is a well recognized and established gold standard to probe studies on drug metabolism, cytotoxicity, genotoxicity, hepatotoxicity and tool for chemical risk assessment in vitro. HepG2 cells are comparable to human hepatocytes for evaluation of CYP1A2, CYP2B6, CYP3A4 induction at mRNA and protein levels credited to their phenotypic stability, high differentiation and immortality advocating their use as a cell based model in drug discovery.

Despite the fact that conventional 2D HepG2 cells have provided a wealth of information, it has been shown in recent reports that such conventional 2D planar HepG2 cell cultures represent disparity to primary hepatocytes in certain structural and functional characteristics. Therefore, three dimensional (3D) human hepatoma cell models are being developed as a resource to overcome functional aberrations associated with the 2D culturing method and to achieve more accuracy in simulating the in vivo tumor microenvironment. In this regard, bioengineered 3D hepatic models provide more realistic microenvironment (biomolecular, biochemical and biomechanical conditions) and maintain the fluidity of model. Overall, 3D platform optimizes predictive value, metabolic stability and reliability to scrutinize molecular response of herbals in drug testing and is clearly expected to be a sought after useful reference for research in hepatoma modeling.

Identification of botanicals employing two dimensional (2D) hepatoma cell cultures

HepG2 mediated bioactivities of herbal plants and their active principles are represented in Table 1. For instance, a cumulative effect of decoction of Nigella sativa seeds, Hemidesmus indicus root, and Smilax glabra rhizome holds traditional repute as a curative measure for cancer and other diseases. These three herbal species account for their cytotoxic action against HepG2 cells via silencing incorporation of [14C]-leucine into protein and [3H]-thymidine into DNA of cells eventually leading to the decreased synthesis of DNA and proteins. This in turn induces cell necrosis and/or apoptosis of hepatic cancer cells. Further studies corroborate anti-hepatocarcinogenic effect of herbal formulation by inducing cell apoptosis. This is characterized by induction of DNA fragmentation, nuclear condensation, membrane blebbing, and formation of apoptotic bodies escorting enhancement in p53, p21, caspase-3, caspase-9, expression of Bcl-2-associated X protein (Bax) and suppression of Bcl-2 (Samarakoon et al. 2012). Huanglian Jiedu formulation has been valued clinically for treatment against HCC. It comprises berberine, geniposide, palmatine, and baicalin as bioactive constituents. This herbal formulation represses nascent protein synthesis in HCC cells by inactivation of eukaryotic elongation factor 2 (eEF2) via activating phosphorylation of elongation factor 2 (EF2) kinase (Wang et al. 2015). The study advocates suppression of proliferation, angiogenesis, and invasion of HCC cells without deregulation of initiation factors in translation. Furthermore, an amalgamation of Curcuma longa rhizome, Piper nigrum fruit, and Allium sativum bulb synchronizes bioenergetic mechanism in HepG2 cells. This synergism of three spices relapse activity of lactate dehydrogenase, elevates pyruvate dehydrogenase level, regulates the expression of p53, MYC mRNAs, HIF-1α, VEGF and thus, revert cancer cells to normal via modification of lactic acid fermentation to oxidative phosphorylation (Ramachandran and Menon 2016). A Japanese–Chinese traditional herbal formula, comprising roots of Scutellaria baicalensis, Coptis japonica, Gardenia jasminoides, and Phellodendron amurense, reveals the deterrent activity of acyl-coenzyme A: cholesterol acyltransferase expression in HepG2 cells emphasizing prominence of formulation in inhibiting lipid biosynthesis and maintenance of cholesterol homeostasis (Yotsumoto et al. 1997). Qushi Huayu decoction hampers triglyceride content, restrains P-IκB and TNF-α expression, thereby slumping secretion of TNF-α and suggests the curative action of this herbal mixture on hepatic steatosis. HPLC analysis of the herbal combination elucidated the presence of chlorogenic acid, resveratrol, jasminoidin and polygonin which might be accountable for its preventive efficacy against hepatic lipotoxicity (Feng et al. 2013). Dai-saiko-to decoction, a combination of seven herbals, reduces the level of VLDL and triglyceride synthesis in plasma and hence exerts hypocholesterolemic potential. Addition of another herbal, i.e., Citrus aurantium to this decoction regulates the level of plasma lipids due to rise in the expression level of LDL receptor and SREBP-2 (Iizuka et al. 2013). A Chinese herbal decoction, Wu-Zi-Yan-Zong-Wan, renders cytoprotection against oxidative injury exaggerated by ethanol in cytochrome P450 2E1 cDNA-transfected HepG2 (E47) cells. This five component decocted extract unveils presence of marker compounds such as betaine, quercetin, schisandrin, hyperin, schisandrin B and deoxyschisandrin as active ingredients. This herbal formula turns down lactate dehydrogenase leakage, reactive oxygen species production, lipid peroxidation and raises content of reduced glutathione, membrane potential, precludes DNA ladder formation and hence affords protection in opposition to oxidative damage (Chen et al. 2010). Ethyl acetate extract of an herbal formula known as Jiedu Xiaozheng Yin decoction has been reported to arrest the growth of HepG2 cells at G0/G1 phase via increasing cyclin D and E expression (Cao et al. 2013). These various combinatory decoction studies indicate that HepG2 cells can be effectively employed to determine their antiproliferative activity by inducing apoptosis and suppressing survival genes such as Bcl-2.

Table 1 HepG2 mediated bioactivities of herbal plants and their active principles
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Experiments conducted by Bhatia et al. (2015) reflects pronounced efficiency of aqueous extract of Origanum dayi and Ochradenus baccatus plants in promoting apoptosis via intrinsic mitochondrial pathway by regulating mRNA expression of P450 enzymes, caspases (3, 7 and 9), Bax, Bcl-2-associated death promoter (Bad) and poly(adenosine diphosphate-ribose) polymerase (PARP). Terminalia arjuna aqueous extract impedes alterations in redox status incited by tertiary butyl hydroperoxide in cultured hepatoma cells via upregulating level of antioxidant enzymes by its bioactive components (Shivananjappa et al. 2013). In a similar trend, aqueous extract of Barringtonia racemosa (leaf and stem) and ethyl acetate extract of Lavandula coronopifolia (aerial parts) sustains cellular homeostasis and grants protection against toxic response initiated by reactive damaging entities (Kong et al. 2016; Farshori et al. 2015). Available literature thus reflects the imperative utility of HepG2 cells to be employed as oxidative stress model as these cells preserve endogenous functions and expression of diverse antioxidative enzymes. HepG2 cells provide an excellent tool for appraising oxidative damages incited by various toxins and to discover vital mechanisms associated with protection presented by numerous botanicals against oxidative insults. Herbal extracts have also been estimated for their anti-insulin resistant action using dexamethasone incited insulin resistant HepG2 cells as an evaluation model. Plants enhance extracellular glucose consumption and reduce intracellular glycogen content underscoring their potential to amplify insulin sensitivity (Ma et al. 2014). These results suggest that HepG2 in 2D conventional culturing conditions can be successfully employed to determine hepatoprotective, antioxidant enzymatic gene expression and redox alterations against oxidative stress. These cells can also be employed for insulin regulation in energy metabolism studies.

There are numerous studies where purified known isolated compounds can also be evaluated in this culture model. For example, pre-exposure of HepG2 cells with 3″-O-methylcrenatoside, leucoseceptoside A, crenatoside, and martynoside isolated from Incarvillea compacta roots fortifies antioxidative enzymatic machinery and downregulates transactivation of NF-κB in luciferase reporter gene assay. Thus these phenylethanoid glycosides operate as hepatoprotectants on carbon tetrachloride aggravated damage in HepG2 cells (Shen et al. 2015). Gardenoside, an extraction product of G. jasminoides, intervenes with hepatic lipotoxicity induced in HepG2 cells by free fatty acid mixture (oleic and palmitic acid mixture, 2:1). This compound effectively attenuates secretion of inflammatory cytokines and averts translocation of NF-κB into nucleus preventing its activation and renders protection against cellular steatosis (Liang et al. 2015). Quercetin also modulates alterations in expression of antioxidant biomarkers in response to oxidative damage aggravated in human hepatoma cells by tert-butyl hydroperoxide (Alia et al. 2006). Fatsioside A (baccharane-type triterpenoid glycoside) extracted from Fatsia japonica fruit activates AMPK signaling, increases acetyl-CoA carboxylase expression and promotes hepatoma cell death (Zheng et al. 2015). A steroidal saponin, known as polyphyllin I extorted from the rhizome of Paris polyphyllin exhibits repression ability against constitutive and cisplatin provoked phosphorylation of p65 protein and c-Myc, Bcl-2 and VEGF expression level (Han et al. 2015). These results indicate dual nature of HepG2 cells, facilitating studies on hepatoprotective action by regulation of NF-κB and anti-hepatoma activities via modulation of VEGF and Bcl-2 expression. Overall, HepG2 mediated cumulative biomodulatory actions of herbal plants and their active principles have been represented in Fig. 1. The figure clearly illustrates the range of 2D HepG2 culturing techniques for evaluation of hepatoprotective and anti-hepatoma studies. 2D model can also be employed for evaluation of cholesterol homeostasis and insulin regulation.

Fig. 1
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HepG2 mediated cumulative biomodulatory actions of herbal plants and their active principles

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Programming of 3D hepatoma cell culture

Foregoing studies on medicinal plants advocate 2D monolayered hepatoma cells as in vitro reference model to determine their initial activities. However, some studies propose that functions and expression of certain genes in HepG2 cells do not resemble primary hepatocytes as former represents feeble expression of cytochrome P450 enzymes and xenobiotic receptors in conventional 2D culturing method (Ramaiahgari et al. 2014). There is rapid dedifferentiation of hepatocytes with degradation in phenotypic characteristics, collapse of biological functionalities such as loss of cell–matrix and cell–cell interactions, poor secretion of plasma proteins and alterations in cell signaling pathways. Thus, HepG2 cells cultured on rigid plastic surfaces have metabolic incompetence to envisage drug-induced hepatic injury.

Keeping in view, problems associated with the 2D cellular model, 3D in vitro culture approaches to cultivate HepG2 cells have been developed. In 3D modeling, cells and their extracellular matrix are considered as functional units to improvise drug development. An extensive source of cells for 3D modeling including stem cells, cell lines, allogenic cells, autologous cells, xenogenic cells, animal derived primary cells, and their genetically modified variants have been employed to generate 3D models. The basic procedure engrosses generation of aggregates or spheroids using scaffolds/matrices, which may be biological or synthetic in nature. Cells are plated on an acellular matrix or dispersed within the matrix and then solidified or polymerized. Other scaffold-free system entails forced floating, hanging or agitation-based methods in which 3D spheroids are grown in suspensions (Edmondson et al. 2014). In addition, these static methods are being integrated with bioreactors to regulate environmental circumstances obligatory for cell cultivation.

In 3D culture study, HepG2 spheroids generated on extracellular matrix loaded hydrogels have been proposed as a suitable model in toxicity and safety evaluation studies. These hepatic spheroids mimic tissues of actual liver structurally and demonstrate elevation in metabolic expression of key regulators viz., phase I enzymes (CYP1A2, CYP3A4, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP4F3), phase II enzymes (UGT1A1, UGT1A3, UGT1A6, UGT2B4, SULT2A1), phase III drug transporters (OAT7, OAT2) and nuclear receptors (CAR, AhR, PXR) involved in drug metabolism for longer duration (Ramaiahgari et al. 2014). However, several small spheroids readily unite to form spheroids of bigger diameter with cellular necrosis in the core due to lack of oxygen and nutrient insufficiency. Therefore, HepG2 cells have been cultivated in micro space culture plates as this methodology is effective in maintaining the diameter of spheroids below the level of survival threshold (Nakamura et al. 2011). These results suggest that 3D HepG2 spheroidal culture can be successfully utilized to determine effects of experimental extract or chemicals on the metabolic profile of hepatoma cells. Further, nano-cultured cells represent even high susceptibility to CYP2E1 expression and this model surrogate mechanistic characteristics, i.e., decline in mitochondrial membrane potential, glutathione content and phosphorylation of JNK associated with paracetamol provoked hepatic damage (Aritomi et al. 2014). In a 2D–3D hybrid system, encapsulation of HepG2 cells in alginate hydrogel propped up by poly-carbonate platform and co-cultured with MCF-7 cells respond with raised sensitivity to acetaminophen, rifampin, diclofenac, and quinidine (Lan and Starly 2011). 3D multicellular heterospheroids prepared in collagen hydrogel culture also demonstrate resistance to doxorubicin over monolayered and homospheroid cells. This might be due to the creation of oxygen gradient in the spheroidal core that replicates hypoxic environment observed within solid tumors and modulates cells to defy diffusion of drugs and thus confirm better viability (Yip and Cho 2013). 3D cell models incorporate varied configuration systems to overcome shortcomings in 2D monolayered cultures. Moreover, these systems require microfabricated 3D scaffolds with complex architecture to replicate in vivo like microenvironment to withstand requisite distribution and circulation of oxygen and other molecules (Derda et al. 2009). Application of scaffolds (natural or synthetic polymers or their admixture) that impersonate extra-cellular matrices in permeability, porosity and mechanical consistency illustrate better biochemical and biophysical interventions for expression of biomarkers (Ravi et al. 2015). HepG2 cells developed via 3D porous polystyrene substrate display lower predisposition towards cell death at higher concentrations of methotrexate (hepatotoxin), increase the formation of bile canaliculi and regulate structural integrity as compared to 2D configuration (Bokhari et al. 2007). However, 3D microfabrication involves a time intense advanced engineering specialization and high cost instruments for accurate outcomes. An affordable approach of stacking hydrogel impregnated chromatography papers for microfabrication of 3D cell cultures can be useful substitute method (Derda et al. 2009). In this method, cells impregnated papers can be stacked on cellulose acetate or poly(dimethylsiloxane) (PDMS) films with altering permeability for oxygen and water. Stacked and destacked layers of impregnated chromatography papers regulate oxygen and nutrient gradient thus, enabling determination of viable cells distribution in three dimension facilitating analysis of molecular and genetic outcomes in better recapitulating in vivo scenario. This modeling technique might be a reasonable approach for exploration of prime areas in tissue engineering and biotechnology.

Recent considerations

An innovative coordination of microphysiological liver system encompassing four categories of human hepatic cells structured into a 3D microfluidic sinusoidal complex to detect mechanism of drug toxicity, based on fluorescence biosensors, has been devised recently (Vernetti et al. 2017). Fabrication of hepatoma spheroidal culture in a microfluidic environment using PDMS decreases the resistance of 5-fluorouracil with an increase in spheroidal size due to the release of death signals such as TNF-α by innermost necrotic cells (Zuchowska et al. 2017). Such cell-on-a chip micro device can be successfully employed to evaluate the anticancer proficiency of new drugs. Another liver-on-a-chip based model of HepG2/C3A spheroids entrapped in methacrylate gelatin hydrogel results in increased expression of ceruloplasmin, transferrin, MRP2, cytokeratin 18, ZO-1 and displays toxic response to acetaminophen with similar in vivo scenario (Bhise et al. 2016). HepG2 cells have been soaked in alginate to fabricate 3D bioprinted mCherry-HepG2 hydrogel structures followed by cross linking with calcium chloride. These newly generated cells hold significant promise in advancing gene expression profiling and put forward alternative innovative options to circumvent surgical complications and donor scarcity (Jeon et al. 2017). Hepatospheroids adhered with hybrid scaffolds are prepared by cross-linking collagen with gum arabic. These non-cellular adherent scaffolds favor cell–cell interaction and formation of spheroids in comparison to cellular matrix substrate. HepG2 cells seeded in collagen–gum arabic hybrid substrates are cultured under microgravity conditions simulated by using rotating bioreactor. Cells cultivated under such conditions promote secretion of urea and albumin. Further, development of biological substitutes would be of high impact for in vitro safety evaluation of drugs and in the engineering of bioartificial hepatic devices (Sarika et al. 2016). HepG2 cylindroids within hollow fibers of polyvinylidene fluoride represent better sensitivity at short exposure duration to lower concentrations of acrylamide and sodium nitrite. The rough inner surface of fibers endorses cells to adhere, propagate and express hepatic genes via micropores that facilitate the exchange of materials between inner hepatic cells and culture media. This study urges high throughput toxicity testing of chemical compounds in food manufacturing and processing industries (Chu et al. 2017). HepG2 cells cultured on a liquid–solid interface (2 days) and afterward on a liquid–gas interface (1 day) in collagen vitrigel membrane chamber comprising of high-density collagen fibrils with continuous oxygenation have been considered as the most suitable model for analyzing absorption, distribution, metabolism, and excretion behavior of chemicals. This model reproduces functional characteristics of hepatocytes along with expression of efflux transporters such as MRP2, MRP3, and MRP4 (Luckert et al. 2017). These results clearly prove that 3D HepG2 model or liver cell on chip model is superior to conventional 2D HepG2 cell culture model to understand the efficacy of drugs on the survival of cancer cells, toxicity, and microenvironment. However, in a recent study, HepG2 cells cultured in 2D monolayers for 21 days asserted similarity to 3D culture system comprising of alvetex scaffolds, matrigel matrix, collagen sandwich in the analysis of biochemical markers, hepato-oriented gene expression profile and cellular morphological features (Zuchowska et al. 2017). The rationale for this pronounced resemblance is that during long cultivation for 21 days, cells revealed primary differentiation due to maturation and secondary differentiation during confluency phase. Thus, protracted culture duration is accountable for better and increased HepG2 metabolic capability regardless of 2D or 3D culture. Overall structural integrity and viability in HepG2 3D cultures increase with better in vivo like expression as shown in Fig. 2, which also illustrates that 3D model articulates better biomolecular expressions when 2D cell culture model is compared to the development of specialized 3D engineering system.

Fig. 2
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HepG2 3D cultures demonstrating better in vivo like expression with the evolution of specialized engineering system

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Future prospects

Bioengineered 3D culture methods are under expansion to address the urgent need of predictive in vitro hepatic model in drug discovery and research. Although considerable interest has been focused in this area employing hydrogels, microfluidics and bioreactors, the major bottleneck is a deficiency of standardized approach. 3D models have high flexibility and can overcome shortcomings observed with other platforms. However, there is a dilemma in replicating in vivo physiological tissue conditions due to variance in tissue origin, tissue–tissue intervention, their response to mechanical and physical microenvironment, the spatiotemporal gradient of oxygen, nutrients, metabolites and other signaling molecules (Duval et al. 2017). Hence, more efforts are desired to develop, optimize and validate cultivation conditions to mimic in vivo tissue scenario more precisely with cost effective strategies. One of the possible solutions to current challenges could be the development of a combinatorial model with an integration of biological, topographical and mechanical factors with the low cost set-up. This could be achieved by use of natural biological substrates to construct a versatile model for diverse cell types. Natural biosubstitutes could steer signaling cascades associated with cancer cells and may offer constructive research outcome that may improve strategies for 3D disease modeling design.

Conclusion

Numerous studies on the investigation of herbal bioactivities have been carried out by using HepG2 cell cultures. However, HepG2 cells in 2D culture conditions have been reported to display inadequate expression of different cytochromes, albumin, and α-1 antitrypsin in comparison to primary human hepatic cells. In this context, it is assumed that more studies on biomimetic 3D hepatoma cell cultures can better reflect their expression to reiterate in vivo tumor microenvironment and can be employed to decipher molecular response of herbal isolates to recognize novel prognostic targets for pre-clinical drug discovery studies. Innovative bioengineered 3D hepatoma models hold promise to identify crucial biomarkers in herbal botanicals associated with different diseases to begin tailoring treatment strategies for individual patients.