Hepatocellular carcinoma (HCC) is the predominant primary cancer in most countries and seventh most frequent cancer across the globe (1). HCC is the second most lethal cancer-associated mortality in the world, with an annual rate of around 1.2 million deaths, mainly in the under-developing and developing countries of East Asia and Africa (2). The incidence of HCC varies according to gender, age, ethnicity and geographical distribution. The majority (more than 80%) of HCC cases occur in African and East Asian countries, with an incidence rate of more than 20 people in 100 000 individuals. HCC rarely appears below the age of 40 and males are at higher risk than females (3). Several etiological factors that have a casual association with HCC are chronic viral infections such as hepatitis B virus (HBV) or hepatitis C virus (HCV), frequent exposure to aflatoxin B1, liver cirrhosis due to alcohol abuse, and non-alcoholic fatty liver (4). Patients with early disease are often asymptomatic and consequently diagnosed at a late stage when the disease is untreatable (5).

At the molecular level, HCC is complex and heterogeneous due to several key mutations. These mutations cause aberrant modulations of key proteins that result in the deregulation of crucial signalling pathways (6, 7). Owing to many genetic mutations in the HCC and the deregulation of many signalling pathways, HCC is often resistant to the current regiment of therapeutic modalities (8). The therapeutic options are surgical resection and are often more successful if the malignancy is diagnosed at an early stage of the disease (9). Although liver transplantation is the best effective therapeutic approach to deal with HCC; only around 5% of patients can benefit from this approach because of histocompatibility and organ availability issues (10). Except transplantation, other therapeutic options are chemotherapy and radiotherapy. Regrettably, HCC resists most cytotoxic drugs. Among chemotherapeutic drugs, sorafenib, a kinase inhibitor, is the drug of choice against HCC and increases patient’s survival time for only a few months (11).

For the last two decades, various novel synthetic chemotherapeutic drugs have been used in clinical settings to treat cancer, but still not much success has been achieved due to considerable cost of development and off-target deleterious effects on normal cells, which causes serious adverse effects like gastrointestinal upset, suppression of immune system, and the development of drug resistance, thereby leading to failure of these drugs (12). Thus, identification and evaluation of plant-based novel extracts or compounds are seemingly important.

Natural products obtained from the plant kingdom have received great attention for promising chemotherapeutic potential and are the prime source of medicines for other ailments. Owing to their structural complexity, natural products can hit multiple targets at various stages of tumourigenesis and associated inflammation (13). This provides evidence that natural products are promising anticancer molecules to target deregulated signalling pathways at multiple sites to curtail cancer. More than 60% of anticancer drugs come from plant-based natural products or their derivatives (14). At present, around 3000 plants across the globe have been reported to have anticancer potential. Globally, around 10 to 40% of plant-derived products are used for the treatment of cancer and are expected to reach 50% in Asia (15). Therefore, there is a continuous demand for search and development of novel and effective drugs from natural sources (plant sources) which specifically kill tumour cells with fewer undesirable effects against normal cells.

Plant-based extracts or natural compounds derived from plant extracts with chemopreventive activity are a vital source of drugs against many diseases including cancer (16). Approximately more than 50% of the current regiment of the Food and Drug Administration (FDA)-approved drugs are derived from plants (17). Yet, there are numerous plants of various species that could have the potential to exhibit pharmacological activities and can be an important source of novel compounds for future therapeutics with great efficacy against tumour cells and fewer toxicity issues against normal human cells. Intending to search for novel compounds with less deleterious effects, we focused on natural medicines. One of the plants from the deserts of Saudi Arabia containing a variety of secondary metabolites that could be used for the production of anticancer therapeutics is Tamarix articulata (18). The plant belongs to the family Tamaricaceae and is a halophytic plant growing in extremely harsh and arid conditions. Traditionally, the plant is used to treat various ailments such as gastrointestinal, skin and heart diseases and other ailments (19). However, recent evidence suggests that owing to the presence of various polyphenolic compounds, T. articulata exhibits some promising pharmacological activities such as antioxidant, antidiabetic, hypolipidemic, antibacterial and hepatoprotective activities (20-24). The major chemical constituents of the T. articulata dry leaf extract are quinic acid, gallic acid, kaempferol, quercetin, tamarixetin, epicatechin gallate and epiafzelechin. These phytochemicals are responsible for pharmacological properties including antioxidant, antibacterial, anticancer, antidiabetics and hepatoprotective activities (18). Crude extracts of the plants in other parts of the world have been reported to exhibit anticancer activities by inhibiting cell viability in various types of cancer cells (17). Our recent study (20) reveals that methanolic extracts of all T. articulata parts (root, stem, dry and fresh leaves) collected from the Qassim region of Saudi Arabia have promising antioxidant activity due to the presence of polyphenolic and flavonoid compounds. Additionally, we documented that all methanolic extracts of different plant parts exhibit potential anticancer effect; however, the data were preliminary and without any mechanistic study. Therefore, in the current study, we evaluated the antiproliferative activity of the methanolic extract of dry leaves of T. articulata against a panel of HCC cells (HepG2, Hep3B and Huh7D12) in an attempt to better understand the underlying anticancer mechanism. We observed that the plant methanolic extract simultaneously induces apoptosis and autophagy in HCC (Hep2G) cells. Additionally, the plant methanolic extract triggered the arrest of the G₀/G₁ phase of the cell cycle and increased the protein expression of cyclin-dependent kinase inhibitor 1B (p27/Kip), encoded by the CDKN1B gene and cyclin-dependent kinase inhibitor 1 (p21/Cip), encoded by the CDKN1A gene, with a decrease in the expression of cyclin D1 in Hep2G cells. Together, these results proved that T. articulata medicinal plant can be explored as a potential drug candidate to curtail cancer in near future.

The current study demonstrated for the first time that the methanolic extract of Tamarix articulata dry leaves exhibits cytotoxic potential and promotes simultaneous induction of autophagy and apoptosis in HCC (HepG2) cells. A mechanistic study reveals that upon treatment with various doses of the plant methanolic extract, HCC (HepG2) cells augment autophagic cell death by promoting the formation of autophagosomes, which were analysed by acridine orange staining. Furthermore, the immunoblotting demonstrates the steady conversion of microtubule-associated protein 1A/1B-light chain 3-I (LC3I) to microtubule-associated protein 1A/1B-light chain 3-II (LC3II) with gradual degradation of sequestosome-1 (SQSTM1/p62) upon treatment with various doses of the plant extract. Moreover, we observed a significant induction of apoptosis, which was supported by activation of apoptosis marker proteins caspase-3 and PARP1 at higher doses of the plant methanolic extract. DAPI staining, phase contrast microscopy and elevated levels of ROS further confirmed the cell killing by apoptosis. Additionally, the plant methanolic extract triggered cell cycle arrest at the G₀/G₁ phase and increased the expression of cell cycle checkpoint proteins p27/Kip and p21/Cip with a decrease in the expression of cyclin D1 in the cells treated with various doses of the plant methanolic extract (Fig. 1). Together these results suggest that the medicinal plant T. articulata can be explored as a potential drug candidate to curtail cancer in the future.

Previous reports demonstrated that numerous plant extracts exhibit an antiproliferative potential against a wide range of cellular models of cancer (33, 34). T. articulata methanolic extract reveals several pharmacological properties like anti-inflammatory, hepatoprotective, antioxidative and antiproliferative activity by modulating miR-1275 in hepatocellular carcinoma cells (18, 20). Thus, we sought to examine the in vitro inhibitory activity of the plant methanolic extract against the HCC cell (HepG2, Huh7, Hep3B and THLE-2) proliferation. We observe that the plant methanolic extract started attenuation of growth kinetics of HCC (HepG2, Huh7, Hep3B and THLE-2) cells at an incredibly low dose of 10 µg/mL. It was observed that the IC₅₀ value was (271.0±4.4), (298.0±7.1) and (336.0±6.1) µg/mL against HepG2, Huh7D12 and Hep3B cell lines, respectively (Fig. 2 and Table 1). Interestingly, when the inhibitory effect on the growth of transformed normal THLE-2 cells was evaluated, we observed that 50% population of the cells died at (3641.0±23.7 µg/mL) of the plant methanolic extract (Fig. 2 and Table 1), which is a significantly high dose of the plant methanolic extract compared to IC₅₀ dose against HCC (HepG2, Huh7, Hep3B and THLE-2) cells. This indicates that the plant methanolic extract not only curtails the cellular growth kinetics of HCC cells specifically, but also displays safe cytotoxicity potential against normal cells. Together, our results suggest that the methanolic extract of dry leaves of halophytic T. articulata plant displays potential cytotoxic effect against a panel of HCC cells and leaving normal transformed liver cells with weaker cytotoxic effect.

Next, the microscopic analysis indicated that a substantial number of dead, floating tumour cells were observed at higher doses of the plant methanolic extract than untreated control (Fig. 3a), indicating that the percentage of cell death in groups treated with the plant extract was significantly higher than with the DMSO control. Furthermore, cell killing by the plant methanolic extract was also confirmed by the nuclear morphology of DAPI stain, which revealed the change in the membrane blebbing and nuclear morphology, which are the hallmarks of apoptosis (Fig. 3b). The above results demonstrate that the plant methanolic extract induces a potent antiproliferative effect on the cellular models of HCC (HepG2, Huh7, Hep3B and THLE-2).

Badmus et al. (35) suggested that Holarrhena floribunda extract displayed an antiproliferative activity against tumour cells and triggered cell cycle arrest by inducing ROS-mediated apoptosis induction. Similarly, Kowalczyk et al. (36) showed that Menyanthes trifoliata L. extract significantly inhibited the growth of glioma cells by attenuating the expression of anti-apoptotic proteins, triggered arrest of the G₂/M phase of cell cycle, activated mitochondrion-dependent apoptosis and augmented the expression of apoptosis proteins Bax and caspase-3. Various studies have revealed that natural compounds obtained from plants have a promising apoptosis-inducing ability in cancer cells (37–39). Mechanistically, apoptosis induced by natural compounds disrupts the association of antiapoptotic protein Bcl-2 from the proapoptotic proteins, thereby activating a cascade of proapoptotic proteins (40). Plant-based extracts and derived compounds have been documented to activate cell death mechanisms by activating cellular caspases, promote intrinsic apoptosis by releasing cytochrome C, induce ROS production and activation of 5' adenosine monophosphate-activated protein kinase (AMPK) which ultimately leads to activation of cell death mechanisms in tumour cells (41, 42). Thus, we wanted to evaluate the apoptotic potential of T. articulata methanolic extract against HCC (HepG2) cells. Therefore, we exposed HepG2 cells to different doses of the plant methanolic extract for 24 h and untreated control. With flow cytometry, we observed that a substantial cellular population of HepG2 cells underwent apoptosis (23.1%) at a higher dose of the plant methanolic extract (500 μg/mL) than with positive control staurosporine (25 nM) (42.1%) (Fig. 4a and Fig. 4b). To further confirm the activation of apoptosis, we performed immunoblotting analysis of HepG2 cells treated with the plant methanolic extract in a concentration-dependent manner (125, 250 and 500 µg/mL). Our results suggest that higher concentrations of the plant methanolic extract activate apoptosis-promoting proteins by inducing prominent cleavage of caspase-3 and PARP1 when compared to untreated control (Fig. 4c).

ROS and associated peroxidases play a key role in the regulation of cell death in multiple cancer cell types. Accumulated evidence revealed that elevation of ROS by natural products is the major driver of the activation of cancer cell death pathways. In the recent past, reports have suggested that chemotherapeutic agents induce apoptotic cell death by elevating ROS production (31). To examine whether the plant methanolic extract could also induce ROS production in HepG2 cells, we exposed HepG2 cells to various doses of the plant extract in the presence of ROS activator H₂O₂ (positive control, 100 μM) or untreated control for 24 h and stained the cells with DCFH-DA dye after the treatment. DCFH-DA dye easily passes through the membrane by diffusion and is deacetylated with cellular esterases to non-fluorescent compound. The non-fluorescent compound is then oxidised to produce green colour compound 2',7'-dichlorofluorescein in the presence of ROS. Intriguingly, we observed that the plant extract promotes significant ROS production in a concentration-dependent manner as compared to untreated control. However, HepG2 cells exposed to positive control (H₂O₂, 100 μM) significantly promoted ROS production (Fig. 5). Together, these results demonstrate that the plant methanolic extract activates apoptosis-mediated proteins and promotes ROS production, which might have a critical role in the augmentation of apoptosis.

Autophagy is one of the important catabolic physiological processes that help clear the old and damaged cellular contents, recycle metabolites with the help of lysosomes and utilize them during energy crisis. It plays a dual role in cancer therapy and tumourigenesis. Accumulating evidence suggests that most natural products that exhibit antiproliferative activity induce autophagic cell death mode in tumour cells (31). A previous report revealed that in hepatocellular carcinoma cells, berberine suppresses hepatocellular carcinoma cells by inducing simultaneously mitochondrion-mediated apoptosis and autophagic cell death (43). Numerous reports suggest that plant-based natural compounds and extracts induce autophagy and kill tumour cells by various cell death mechanisms (44, 45). Therefore, we sought to reveal whether T. articulata methanolic extract treatment could augment the induction of autophagy in HepG2 cells. To evaluate the effect of T. articulata methanolic extract, the HepG2 cells were plated overnight and exposed to various doses of the plant extract (Fig. 6a). After the completion of 24-hour incubation, the cells were stained with acridine orange. It is observable in Fig. 6b and Fig. 6c that although majority of the cells exposed to lower doses of the plant methanolic extract could not promote autophagy significantly and displayed acidic vesicular organelles (AVOs) in the cytoplasm, cells exposed to higher doses of the extract significantly induced AVOs, which appear bright red in the cytoplasm of HepG2 cells. Furthermore, immunoblotting of HepG2 cells at higher doses of the plant methanolic extract revealed a prominent conversion of LC3I to LC3II which indicates the maturation of autophagosome and induction of autophagy (Fig. 6c). To further evaluate the effect of the plant methanolic extract on the flux of autophagy in HepG2 cells, we observed the reduced expression with the low band intensity of sequestrosome 1 (P62) protein, which is a specific substrate of autophagy, at higher doses of the plant methanolic extract than at lower doses. Collectively, these results suggest that HepG2 cells exposed to the plant methanolic extract stimulate autophagic flux and augment cell death in tumour cells.

Accumulating evidence suggests that numerous phytochemicals derived from natural sources have been reported to exhibit antiproliferative activity by triggering cell cycle arrest, thereby promoting cell death via apoptosis (45, 46). Tumour cell proliferation is primarily controlled by the cell cycle (46), which consists of four distinct progressive phases (subG1/G0, G1, S and G2/M) (47). Under normal circumstances, the progression of the cell cycle is tightly regulated by an activation cascade of cyclins, cyclin-dependent kinases (CDKs), and CDK inhibitors. However, in malignant cells, aberrant expression of cell cycle regulators attenuates the differentiation of cells that results in irregular cell growth. In the recent past, numerous reports have suggested that natural compounds have antiproliferative activity by triggering cell cycle arrest (48). Luo et al. (49) demonstrated that 6-gingerol (6-G) enhances the radiosensitivity of gastric cancer cells by the induction of apoptosis and cell cycle arrest. Additionally, Lin et al. (50) revealed that 6-G suppresses cell motility of colon cancer cells by inducing ROS, and upregulates p21/Cip and p27/Kip, thereby arresting cell cycle in colon cancer cells (50). To examine whether T. articulata methanolic extract could arrest the cell cycle, HepG2 cells were synchronised in a low serum-containing medium, and then advanced through various cell cycle phases in a serum-containing medium for at least 3 h. The cells were then treated with various doses (125, 250 and 500 µg/mL) of the plant methanolic extract for 24 h. Our results demonstrated that T. articulata methanolic extract arrested the cell cycle at G₀/G₁ phase in HepG2 cells. The quantification of cells at various cell cycle phases showed that a significant growth of cells (18.1%) was halted at the G₀/G₁ phase of the cell cycle when HepG2 cells were exposed to 500 µg/mL of plant extract as compared to untreated control (3.6%) and positive control 2 µM camptothecin (25.4%) (Fig. 7a and Fig. 7b). Next, we sought to examine the effect of the plant methanolic extract in the cell cycle regulatory proteins. Our immunoblotting results demonstrate a drastic reduction in cyclin D1 expression and a subsequent increase in the expression of p21/cip1 and p27/Kip1 proteins when HepG2 cells were exposed to higher doses of the plant methanolic extract (Fig. 7c). Collectively these results demonstrate that the plant methanolic extract induced G₀/G₁ phase cell cycle arrest and increased the expression of cell cycle checkpoint proteins p27/Kip1 and p21/Cip1 with a concomitant decrease in the expression of cyclin D1 in HepG2 cells.

Our results document for the first time that the methanolic extract of dry leaves of Tamarix articulata exhibits cytotoxic activity and simultaneous activation of two cell death-associated processes in HepG2 cells: apoptosis and autophagy. Mechanistically, T. articulata induced autophagy, which was confirmed by prominent orange red colour of acidic autophagosomes and steady conversion of LC3I to LC3II with gradual degradation of SQSTM1/p62. Furthermore, the plant methanolic extract induced ROS-mediated apoptosis, which was confirmed by the activation of apoptosis marker proteins caspase-3 and PARP1. Additionally, the plant methanolic extract triggered the arrest of the G₀/G₁ phase in the cell cycle and increased the expression of cell cycle checkpoint proteins p27/Kip and p21/Cip with a decrease in the expression of cyclin D1 in the cells treated with various doses of plant methanolic extract. Collectively, these results prove that the medicinal plant T. articulata can be evaluated further as a prospective drug candidate compound to inhibit cancer.