Hormetic effect of an Ethanolic Graviola Leaf Extract on HGF-1 cells survival

Abstract

Plant-extracted compounds have been used for centuries in traditional pharmacopeia. Some of them have proven to be excellent drug alternatives for cancer treatment as they target metabolic pathways that are key to cancer cells such as apoptosis, energy-producing catabolic pathways, and the response to oxidative stress. Since some anticancer drugs have been shown to produce dose dependent biologically opposite effects, it is crucial to determine the range of doses for which the compounds have maximum therapeutic benefits. Annona muricata or Graviola is a tropical tree that is common in the Puerto Rican landscape. Although a plethora of studies conducted in vitro and in vivo studies have indeed reported that extracts prepared from the Graviola root, fruit, bark, and leaves possess antiproliferative activities in a large variety of cancer cells, the efficiency of Graviola extracts to curb the progression of head and neck cancers has been overlooked. Furthermore, the bioactivity of Graviola extracts on sane/non-cancerous cells has largely been ignored. The present work reports the in vitro antiproliferative/anticancer behavior of an ethanolic Graviola leaf extract on squamous cell carcinoma cell lines 9 and 25 vs. a sane/non-cancerous human gingival fibroblast cell line-1. Our results show that the Graviola extract induces cell death in the squamous cell carcinoma cell lines at all concentrations tested and a dose-dependent biphasic concentration-dependent/hormetic effect on the fibroblastic cells. This suggests that, at low doses, the phytochemicals present in the prepared Graviola extract could offer potential therapeutic avenues for curbing the progression of head and neck cancers.

Summary:

While the antiproliferative properties of molecules extracted from the tropical tree Graviola have been established in a large variety of cancer cells, the study of the effect of Graviola extracts on models for head and neck cancers have been scarce. Furthermore, the effect of Graviola on noncancerous cells has been barely studied. To fill these knowledge gaps, we conducted an in vitro dose-response experiment of an Ethanolic Graviola Leaf Extract (EGLE) on tongue squamous cell carcinoma (SCC-9 and SCC-25) vs. gingival fibroblasts (HGF-1) cell lines. Our results show a differential survival behavior in the SCC vs HGF-1 cells. While EGLE exerts toxicity to the cancer cells at all the tested concentrations, it, however, induces a dose-dependent biphasic concentration-dependent/hormetic effect on the HGF-1 cells. At doses inferior to 10 ug/mL, EGLE proves to be toxic to SCC-9 and SCC-25 while it enhances the proliferation of the HGF-1, a property that could offer potential therapeutic avenues for curbing the progression of oral cancers. Although the molecular mechanisms accounting for the biphasic effect of the phytoextract on HGF-1 remain to be elucidated, we suspect the hormetic properties of EGLE are correlated to the oxidative status of the HGF-1 cells.

Introduction

One of the characteristics of cancer cells is their high proliferation rate. The drugs used in conventional cancer treatments are antiproliferative agents but unfortunately, they usually offer poor specificity to cancer cells. Natural products, however, and in particular plant-extracted molecules represent excellent alternatives or adjuvants to the conventional anti-tumor treatments because they tend to alter several signaling pathways which are key to cancer progression: among other key metabolic characteristics of cancer cells are accelerated energy production the activation of the oxidative stress response, and inhibition of apoptosis [1].

Annona muricata, also known as Graviola or guanábana is a tropical evergreen tree that is a member of the Annonaceae family. Graviola trees are very common in the Puerto Rican landscape and while Graviola has been used in the Caribbean to cure a large set of ailments, the scientific community has been particularly interested in studying its anti-cancer properties. While decoctions of various parts of Graviola are widely used in ethnomedicinal practices to treat multiple ailments and diseases, the antiproliferative properties of Graviola leaves, bark, fruit, and seed have been reported in vitro and animal models for cancer [2,3]. Furthermore, over 200 bioactive compounds some of which with antiproliferative activities have been characterized and isolated from Graviola extracts [3,4].

Head and Neck Cancers (HNCs) are a group of cancers that start in the mouth, nose, throat, larynx, sinuses, or salivary glands. HNCs account for approximately 6% of all cancers in the United States. Heavy tobacco users have a 5-to 25-fold higher risk of developing this type of cancer than nonsmokers, and alcohol can further increase this risk. In this study, we have assessed the antiproliferative properties of Graviola Ethanolic leaf extracts (EGLE) in an in vitro model of Head and Neck Squamous Cell Carcinoma (HNSCC), a type of tumor that represents over 90% of all the HNCs.

Our results suggest that the chemical compounds present in EGLE induce biologically opposite effects on HGF-1 cells at different concentrations, a phenomenon that has previously been observed for various phytocompounds and described as the Biphasic Concentration-Dependent Effect (BDCE) or hormetic effect (for a review on phytochemically-induced BDCE, see [5]).

Materials and methods

Collection of plant samples:

About 5 kg of leaves from an Annona muricata (Graviola) specimen tree in Boquerón, Cabo Rojo, Puerto Rico (coordinates: 18 ° 05’11.9 “N 67 ° 08’44.6” W) were collected and processed as described by Velázquez & Cassé (2020). A voucher was deposited at the Mayagüez herbarium of the University of Puerto Rico (Voucher: MAPR, Céline Cassé, 2). The leaves were kept at 4°C for preservation. They were first washed with running water and soap, then, with 4% sodium hypochlorite and deionized water, dried, and cut into small pieces. The samples were then “freeze-dried” (Labconco / Lyph Lock 4.5 lyophilization system, Labconco Corp. TM, Kansas City, MO), milled, and vacuum-stored at −80°C.

EGLE Preparation:

EGLE was prepared as follows: 5 g of pulverized Graviola leaves were macerated at room temperature in 50 mL of pure Ethyl Alcohol-200 anhydrous (Sigma Aldrich, St Louis, MO) for 42 hours. The resulting mixture was vacuum filtered (qualitative filter paper, grade # 415, VWR TM, Radnor, PA). The solid parts were discarded, and the filtrate was then

Solubility assays:

The solubility of EGLE in cell culture media was verified by following the protocol described in Velázquez and Cassé [6]. Briefly, 10%, 1%, and 0.1% (v:v) serial dilutions of EGLE were prepared in distilled water. The dilution samples were then centrifuged at 5,000 rpm for 5 minutes. The presence of a pellet corresponding to the insoluble components of the EGLE was visually assessed.

Dose-response analyses:

The tongue SCC-9, SCC-25, and gingival HGF-1 cells were obtained commercially from the ATCC company ((#CRL-1629TM, #CRL-1628TM, #CRL-2014TM), ATCC, Inc., Manassas, VA, USA). The cells were grown in vitro according to the manufacturer’s protocol. The cell culture medium consisted of a 1:1 mixture of Medium Dulbecco’s modified Eagle’s medium and Ham’s F12 medium (DMEM: F-12). The F-12 media contains 1.2 g/L of sodium bicarbonate, 2.5 mM of L-glutamine, 15 mM of HEPES, and 0.5 mM of sodium pyruvate (#ATCC^® 30-2006TM ATCC. Inc., Manassas, VA, USA). It was supplemented with 400 ng/ml of hydrocortisone, 90%, and FBS 10%. The base media for HGF-1 was a formulated ATCC Dulbecco’s Modified Eagle Medium (DMEM), (# 30 2002, ATCC. Inc., Manassas, VA, USA). DMEM was complemented with Fetal Bovine Serum (FBS) at a final concentration of 10%. The SCC-9, SCC-25, and HGF-1 cell lines were cultivated as described according to the manufacturer’s protocol. For the viability assays, 3.0 x 105 cells/ mL of SCC-25, SCC-9, and 1.5 x 105 cells/ mL of HGF-1 were seeded into individual wells of a 96-well microtiter tissue culture plate. After 24 hrs of incubation, the original growth medium was removed and replaced with an exposure medium, containing the serial dilutions of EGLE for 24 hrs. In addition to the standard points, the experiments included a blank control consisting of cell culture medium alone (BC), untreated cells (NC), and solvent control EtOH 0.3% (v:v) (EC) in cell media. Three replicates were run for each EGLE concentration. After a 24-hour incubation, cell viability was assessed by the MTT colorimetric assay. Individual wells of a 96-well microtiter tissue culture plate were seeded with 100 ul of serial 2-fold-dilutions of SCC-9 or SCC-25 at concentrations ranging from 1.95 x 103 to 106 cells/mL, corresponding to 1.95 x 102 to 105 cells/well the absorbance of empty wells was measured as 0 and the background signal from the medium alone was subtracted from each sample measure. Untreated cells were used as the negative control for the experiment. The cells were incubated for 24 hours at 37°C and 5% CO2. After 24 hours of incubation, cell viability was assessed by using the (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide viability (MTT) assays kits (BioVision, Inc. K299-100, SF, for SCC-9; Roche #11465007001, Basel, Switzerland, for SCC-9) cells following the manufacturer’s protocols. The absorbances were measured at OD595 nm using a microplate reader (model MR9600, Accuris Smart Reader TM 96 Microplate Reader, New York, NY).

The percentage of cell survival/viability was calculated using the following equation:

$$\% \hspace{0.17em}\hspace{0.17em}of\hspace{0.17em}cell viability=\left[\frac{\left(As-A_{BC}\right)}{\left(Ac-A_{BC}\right)}\right]\times 100$$ (1)

Where As = Absorbance of Sample, A_BC =Absorbance of Blank, Ac=Absorbancece of Control (no exposure).

The results were averaged and reported as mean ± Standard Deviation (SD). The data for each cell line were plotted using Microsoft Office Excel (Microsoft 365^® Suite, Excel^®, version 16.43).

Results and discussion

To assess if EGLE could qualify as a good in vitro anti-cancer agent, we have compared the antiproliferative properties of the extract on the SCC-9, SCC-25, and HGF-1 cell lines. The effect of EGLE on cell survival was assessed by MTT colorimetric assay, according to the protocol described in the Materials and Methods section and the % of cell survival/viability was calculated by using equation (1). The graphical representation of cell survival percentage is represented in Figure 1.

Figure 1.

Percent of cell survival after a 24h-treatment with EGLE.

The results of the dose-response experiment suggest that for [EGLE]<150 ug/mL, EGLE exerts a dose-dependent antiproliferative effect on the SCC-9 and SCC-25 cancer cell lines at all the concentrations tested.

Furthermore, the antiproliferative effect of EGLE on SCCs is cell-dependent: for [EGLE]< 150 ug/mL, the extract exerts a milder antiproliferative effect on the SCC-9 cells than on SCC-25. This finding is consistent with a previous report by Du et al. which showed that SCC-9 cells possess poor sensitivity to the anticancer/ antiproliferative compound doxorubicin. The sensitivity of the SCC-9 cell line to doxorubicin is believed to be modulated by the upregulated expression of MicroRNA miR-221. miR-221 controls the expression of tissue inhibitor of metalloproteinase-3 (TIMP3), an inhibitor of matrix metalloproteinases involved in extracellular matrix degradation and apoptosis [7].

Contrary to the monophasic behavior of the SCC cell lines, the HGF-1 cells exhibit a dose dependent biphasic response. For [EGLE] < 10 ug/mL, the extract exerts a proliferative effect which is maximum at [EGLE]= 2.3 ug/mL. At such a concentration, HGF-1 cell survival is enhanced by 20% compared to the untreated control. For [EGLE] > 10ug/mL, however, EGLE induces cell death in HGF-1 (% of survival < 100 %). From these observations, we conclude that EGLE exerts a Biphasic Concentration-Dependent (BCDE)/ hormetic effect on the HGF-1 cells.

The present study suggests that some phytocompounds in EGLE exert a hormetic effect on the HGF-1 cells. This finding is consistent with previous in vitro studies of phytochemicals inducing a hormetic response in cancer as well as non-cancer cells. Such molecules include members of the phytoestrogen family, resveratrol, curcumin, sulforaphane, berberine, falcarinol, rutin, and rosmarinic acid, among others [5]. Preliminary chemical characterization we conducted on EGLE concluded of the presence molecules with reported antioxidant, anti-inflammatory, and antiproliferative activities, such as phenolic compounds, flavonoids, alkaloids, terpenoids, and tannins in the extract (data not shown). Furthermore, the antiproliferative/ anticancer properties of the Graviola tree have also been correlated with the bioactivity of annonaceous acetogenins (ACGs), a family of secondary metabolites that have been shown to inhibit Adenosine Triphosphate (ATP) synthesis by targeting the activity of the mitochondrial energy transport chain respiratory complex I/NADPH oxidase complex (NOX) [1,8]. Structurally speaking, ACGs are a series of C-35/C-37 compounds derived from C-32/C-34 fatty acids combined with a 2-propanol unit. ACGs are characterized by a long aliphatic chain bearing a terminal methyl-substituted R alpha, beta-unsaturated gammalactone ring (sometimes rearranged to a ketolactone), with one, two, or three Tetrahydrofurans (THFs) rings located along the hydrocarbon chain, double bonds, and several oxygenated moieties (hydroxyls, acetoxyls, ketones, epoxides). Tetrahydropyran (THP) rings and acyclic compounds are also found in some ACGs [9].

Since the NOX activity is enhanced in cancer compared to sane/non-cancer cells, drugs that target the NOX complex are considered promising anti-cancer agents [10,11]. NOX activates the cell oxidative stress response by producing reactive oxygen species (ROS). NOX-generated ROS are key elements in the maintenance of malignant cells in the sense that ROS act as secondary messengers and have been shown to enhance the activity of oncogenes (Src and Ras) while inhibiting the activity of tumor suppressor genes (p53, PTEN, TSC2). Accelerated glucose uptake and glycolysis are key hallmarks of cancer and NOX also indirectly favor tumorigenicity by activating and stabilizing the subunit-alfa of the hypoxia-inducible factor-1 (HIF1-alpha) which in turn upregulates glycolytic enzymes and glucose transporters [1].

One of the current hypotheses accounting for the BCDE/ hormetic effect of a compound is its modulatory effect on the intracellular ROS levels: low concentrations of drugs are associated with low ROS levels (mild oxidative stress) and higher concentrations of drugs are associated with high ROS levels (strong oxidative stress). In 2019 in vitro study, Posadino et al. have evidenced a BCDE of the polyphenolic antioxidant drug resveratrol on the proliferation of Human umbilical vein endothelial cells (HUVECs). The authors show that at a low concentration (1 uM), resveratrol exerts a proliferative effect on HUVECs. This proliferative effect is correlated with low intracellular ROS levels, cell cycle progression, and activation of the Protein Kinase C (PKC). An opposite antiproliferative effect is, however, observed when the cells are treated with higher doses of resveratrol (10–50 uM), a phenomenon which the authors correlate with a decrease in the level of intracellular ROS, a decrease in the activity of PKC, and activation of apoptosis [12]. Likewise, we make the hypothesis that the BDCE properties of EGLE are correlated to the ROS-dependent oxidative status of the HGF-1 cells: EGLE treatment would enhance the production of intracellular ROS in HGF-1 in a dose-dependent manner. At low concentrations ([EGLE]<10 ug/mL) EGLE would act as a mild oxidant, resulting in a low intracellular ROS and, as a result, the HGF-1cells would proliferate. Moderate to high EGLE concentrations of EGLE (10 ug/mL<[EGLE]<300 ug/mL) would, in turn, result in an overproduction of ROS which would induce apoptosis, hence the antiproliferative effect of EGLE for doses superior to 10 ug/mL.

Oxidative stress is one of the key features of cancer and cancer cells have higher levels of intracellular ROS than sane/non-cancer cells. On the other hand, overproduction of intracellular ROS induces apoptosis in cancer cells, and triggering apoptosis by increasing the level of ROS in the diseased cells has been one of the strategies for eliminating cancer cells [13]. To account for the antiproliferative property of EGLE on SCC-9 and 25, we make the following hypothesis: EGLE treatment further increases the already high level of intracellular ROS in the SCC cells. ROS are overproduced which results in cell death by apoptosis.

One of the possible hypotheses accounting for the differential response of cancer vs. non-cancer cells toward low doses of EGLE is that the effect of the extract would depend on the cells’ respective oxidative status: in cancer cells, with high intracellular ROS levels (SCC-9, SCC-25) low doses of EGLE would induce apoptosis, and non-cancer cells with low ROS levels (HGF-1) would proliferate.

The results of the present study suggest that at low doses, phytochemicals present in EGLE could serve as excellent anticancer drugs since they enhance the proliferation in sane cells while presenting toxicity toward the SCC cells. This is a promising observation with potential therapeutic implications for curbing the progression of HNSCC. Further studies are needed, however, to a) Elucidate the molecular mechanisms associated with the hormetic behavior of EGLE and in particular the effect of EGLE on the oxidative status of HGF-1, and b) Determine if the observed effects of EGLE could be extrapolated to other cell lines and animal models for HNSCC.

Abbreviations:

As

Absorbance of Sample

ABC

Absorbance of Blank

Ac

Absorbance of Control

ACGs

Annonaceous Acetogenins

ATP

Adenosine Triphosphate

BCDE

Biphasic Concentration-Dependent Effect

EGLE

Ethanolic Graviola Leaf Extract

EtOH

Ethanol

HUVECS

Human Umbilical Vein Endothelial Cells

Mir-221

MicroRNA-221

NOX

NADPH Oxidase

HGF-1

Human Gingival Fibroblasts Line-1

HNC

Head And Neck Cancer

HNSCC

Head And Neck Squamous Cell Carcinoma

MTT

(3-[4,5-Dimethylthiazol-2-Yl]-2,5 Diphenyl Tetrazolium Bromide)

PTEN

Phosphatase and Tensin Homolog

ROS

Reactive Oxygen Species

SCC

Squamous Cell Carcinoma

SCC-9

Squamous Cell Carcinoma Line-9