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. 2023 Jul 28;8(31):28563–28576. doi: 10.1021/acsomega.3c02953

Hypophyllanthin and Phyllanthin from Phyllanthus niruri Synergize Doxorubicin Anticancer Properties against Resistant Breast Cancer Cells

Ola E Abdel-Sattar †, Rasha M Allam ‡, Ahmed M Al-Abd ‡, Ali M El-Halawany †, Ahmed M EL-Desoky §, Shanaz O Mohamed ∥, Sherouk Hussein Sweilam ⊥,#, Mohammad Khalid ⊥, Essam Abdel-Sattar †,*, Meselhy R Meselhy †
PMCID: PMC10413485  PMID: 37576627

Abstract

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Doxorubicin (DOX) is a cornerstone chemotherapeutic agent for the treatment of several malignancies such as breast cancer; however, its activity is ameliorated by the development of a resistant phenotype. Phyllanthus species have been studied previously for their potential anticancer properties. The current work is aimed to study the potential cytotoxicity and chemomodulatory effects of hypophyllanthin (PN4) and phyllanthin (PN5) isolated from Phyllanthus niruri to DOX against the adriamycin multidrug-resistant breast cancer cells (MCF-7ADR) and elucidate their mechanism of action. The major compounds of the active methylene chloride fraction were isolated and assessed for their potential cytotoxicity and chemomodulatory effects on DOX against naïve (MCF-7) and resistant breast (MCF-7ADR) cancer cells. The mechanism of action of both compounds in terms of their impacts on programmed/non-programmed cell death (apoptosis and autophagy/necrosis), cell cycle progression/arrest, and tumor cell migration/invasion was investigated. Both compounds PN4 and PN5 showed a moderate but similar potency against MCF-7 as well as MCF-7ADR and significantly synergized DOX-induced anticancer properties against MCF-7ADR. The chemomodulatory effect of both compounds to DOX was found to be via potentiating DOX-induced cell cycle interference and apoptosis induction. It was found that PN4 and PN5 blocked the apoptosis-escape autophagy pathway in MCF-7ADR. On the molecular level, both compounds interfered with SIRT1 expression and consequently suppressed Akt phosphorylation, and PN5 blocked apoptosis escape. Furthermore, PN4 and PN5 showed promising antimigratory and anti-invasive effects against MCF-7ADR, as confirmed by suppression of N-cadherin/β-catenin expression. In conclusion, for the first time, hypophyllanthin and phyllanthin isolated from P. niruri showed promising chemomodulatory effects to the DOX-induced chemotherapeutic activity against MCF-7ADR. Both compounds significantly synergized DOX-induced anticancer properties against MCF-7ADR. This enhanced activity was explained by further promoting DOX-induced apoptosis and suppressing the apoptosis-escape autophagy feature of the resistant breast cancer cells. Both compounds (hypophyllanthin and phyllanthin) interfered with the SIRT1/Akt pathway and suppressed the N-cadherin/β-catenin axis, confirming the observed antiproliferative, cytotoxic, and anti-invasive effects of hypophyllanthin and phyllanthin.

1. Introduction

Breast cancer is the most common cancer among women worldwide and represents the leading cause of cancer-related deaths.1 Chemotherapy is a major strategy for the treatment of breast cancer; however, the development of chemoresistance and tumor recurrence remains a common cause of treatment failure and poor clinical outcomes.2,3 Indeed, for a standard chemotherapy drug such as doxorubicin (DOX), patients with breast cancer may develop chemoresistance within only 6–10 months. Moreover, about 30–50% of metastatic breast cancer patients are irresponsive to the DOX treatment. Therefore, targeting drug resistance in breast cancer is crucial.4

Mechanisms of DOX resistance are multifaceted and include upregulation of drug-resistant proteins, changes in membrane permeability of cancer cells, impairment of DNA damage repair mechanisms, and autophagy-arbitrated drug resistance.5 Among these mechanisms, autophagy-mediated chemotherapy resistance has gained increasing attention. Tumor cells can evade apoptosis through autophagy regulation, thereby increasing drug resistance and enhancing tumor survival.6

Sirtuin 1 (SIRT1) has been revealed to control diverse physiological and pathological processes, including autoimmunity, cellular metabolism, and tumor development.7 Studies demonstrated that high expression of SIRT1 significantly promotes breast cancer progression and is associated with DOX resistance via abnormal activation (deacetylation) of several oncogenic pathways such as Akt.8 Thus, targeting the SIRT1/Akt signaling axis represents a cornerstone in the response of breast cancer patients to DOX treatment.9

In addition, SIRT1 induces tumor invasion by targeting epithelial–mesenchymal transition (EMT)-related pathways.10 Over the past decade, an increasing body of evidence indicated that the EMT might be a key mechanism in tumor progression, invasion, metastasis, and DOX resistance as well.11,12 In aggressive breast cancer conditions, cell polarity and cell–cell contact are fallen in epithelial cells, and specific epithelial markers (such as E-cadherin and cytokeratin) are diminished, while mesenchymal characteristics (such as increased cell migration, activation of β-catenin signaling, vimentin, and N-cadherin) are expanded, participating in tumor resistance to chemotherapeutic drugs.13,14 Therefore, targeting EMT is becoming one of the promising strategies for drug resistance reversal in breast cancer.15

Plant-derived phytochemicals have been widely investigated for their therapeutic potential in the prevention and treatment of cancer, besides their established role in chemosensitizing tumors to overcome drug resistance.16Phyllanthus niruri L. (Syn Phyllanthus amarus Schum & Thonn, family Phyllanthaceae) is a small herb widely distributed in tropical areas worldwide (such as India, China, Java, Southern Florida, and Bahamas), extensively used in ethnopharmacology for the treatment of diabetes, hypertension, and jaundice.17 Recently, researchers have shown a growing interest in evaluating the anticancer activity of this herb.18 The whole plant has diverse biological activities such as anti-inflammatory, antitumor, antinociceptive, and antioxidant properties.19 The aqueous extract of the aerial parts of P. niruri showed an antiproliferative effect on human hepatocellular carcinoma cells (HepG2 and Huh-7) and colorectal carcinoma cells (HT-29).20 However, many questions are still pending about the efficacy of this herb in eliminating cancer cells, especially high resistance and recurrence breast cancer phenotype.

In our previous work, lignan-rich extract from the aerial parts of P. niruri was prepared using non-conventional methods to increase the level of lignans calculated as phyllanthin.19 Recently, we investigated the cytotoxic and chemomodulatory effects of crude methanolic extract and several subfractions of P. niruri against MCF-7 and MCF-7ADR breast cancer cells.21 The methylene chloride fraction (CH2Cl2) showed the most potent cytotoxic activity among all tested fractions. Surprisingly, CH2Cl2 was more cytotoxic against the adriamycin (ADR) multidrug-resistant breast cancer cells (MCF-7ADR) when compared to the naïve cells (MCF-7). In addition, this fraction at sub-cytotoxic concentrations significantly enhanced the cytotoxicity of DOX against both the naïve (MCF-7) and the resistant (MCF-7ADR) breast cancer cells. Therefore, the major compounds from this fraction were isolated and assessed for their potential cytotoxicity and chemomodulatory effects on DOX against naïve and resistant breast cancer cells. Also, we investigated the mechanism of action of these compounds in terms of their impacts on programmed/non-programmed cell death (apoptosis and autophagy/necrosis), cell cycle progression/arrest, and tumor cell migration/invasion.

2. Materials and Methods

2.1. Chemicals

Phyllanthin was purchased from Fluka (Lot #BCBL2476V, product of India). The analytical grade solvents used in extraction and chromatographic separation were purchased from El Gomhouria for Drugs Co. (Cairo, Egypt). Solvents of HPLC grade were purchased from Sigma-Aldrich (Steinheim, Germany). Precoated TLC silica gel F254 plates (20 × 20 cm) and RP silica gel were obtained from Sigma-Aldrich chemicals (Darmstadt, Germany). Sulforhodamine-B (SRB) and DOX were purchased from Sigma Chemical Co. (St. Louis, MO, USA).

2.2. Plant Material

Samples of aerial parts of P. niruri L. were collected in August 2018, from Tasek Gelugor, Penang, Malaysia. The plant material was supplied and identified by the staff member of the Malaysian Agricultural Research and Development Institute (MARDI). A voucher specimen (PN-01-082018B) was kept at the herbarium of the Faculty of Pharmacy, Cairo University. The aerial parts of the collected plant were dried and powdered and saved in tightly closed containers until use.

2.3. Extraction and Isolation of Bioactive Compounds

The powdered aerial parts of P. niruri (750 g) were exhaustively extracted with MeOH (3 × 1.5 L). The solvent was removed under vacuum at a temperature not exceeding 60 °C to yield 93 g of MeOH extract (Ext-1). Part of the MeOH extract (70 g) was suspended in H2O (600 mL) and extracted with CH2Cl2 (3 × 400 mL). The solvent was removed by distillation to yield CH2Cl2 fr. (24.5 g). The CH2Cl2 fraction (10 g) was applied on a Si gel column (5 × 25 cm, 230–400 mesh size). Elution was started with 10% EtOAc in n-hexane (500 mL), followed by increasing the percentage of EtOAc up to 50% EtOAc, and the column was finally washed with 100% EtOAc. Fractions (100 mL each) were collected and monitored by TLC. Fractions with a similar chromatographic pattern were pooled together to give 10 major fractions (Frs I–X). Based on TLC and HPLC chromatograms, lignans were eluted in fractions VI–X.19

Fraction III (0.358 g) on crystallization from MeOH yielded compound PN-1 as a white amorphous powder (65 mg) and was identified as β-sitosterol by comparing to the reference sample.22

Fraction VI (0.207 g) was subjected to MPLC on a RP18 column (12 × 2 cm, 40–60 μm, Merck, Germany) using MeOH-0.1% TFA in water (75:25 v/v) as an eluent (flow rate 4 mL/min and 5 mL fractions) to give compound PN-2 as an amorphous powder (53 mg). Similarly, MPLC of fraction VII (230 mg) was treated similarly to give 80 fractions. Frs 30–60 (75 mg) was subjected to HPLC separation on a RP-18 column (Kromasil 100-5-C18, 10 × 250 mm, 5 μm) using a mobile phase composed of MeOH (solvent A) and 0.1% TFA in H2O (solvent B) in the following gradient elution mode: 0 min 0% A, 10 min to 50% A, 10 min to 80% A, 5 min to 85% A, 10 min 85% A, and finally 5 min to 100% A. The condition of chromatographic separation is as follows: flow rate of 2 mL/min, detection at UV 320, 254, and 230 nm, and injection volume was 5 mg/50 μL. Compound PN-3 (tR 30.5 min) was obtained as an amorphous powder (11 mg).

Fraction VIII (144 mg) was subjected to MPLC on a RP18 column (12 × 2 cm) using MeOH-0.05% TFA in water (85:15 v/v, flow rate 5 mL/min), and 30 fractions were collected (4 mL each). Fractions 6–8 yielded compound PN-4 as an amorphous powder (7.5 mg) at tR 10.073 min.

Fraction IX (526 mg) was chromatographed on normal a Si gel column (20 × 3 cm, mesh size 230–400) using 20% EtOAc in n-hexane to give compound PN-5 as an amorphous powder (90 mg) tR 11.46 min.

2.4. Cell Culture

The human breast cancer cell line (MCF-7) and the DOX-resistant breast cancer cell line (MCF-7ADR) were obtained from Nawah Scientific Inc. (Mokattam, Cairo, Egypt) and cultured in Dulbecco’s modified Eagle’s medium, Gibco-Thermo Fisher Scientific, Waltham, MA, USA. The culture media were complemented with 10% FBS (fetal bovine serum) and 100 units/mL PS (penicillin/streptomycin). The cells were passaged at 37 °C in a humidified atmosphere with 5% CO2.

2.5. Cell Viability Assay

The cell viability was determined by the SRB assay. MCF-7 and MCF-7ADR cells were seeded in 96-well plates, approximately 104 cells/well. After treatment for 72 h, the media were replaced by 150 μL of 10% TCA (trichloroacetic acid) (Merck) for 1 h at 4 °C, followed by five times washing with distilled water. Afterward, 70 μL of SRB solution (0.4% w/v) (Sigma-Aldrich) was added for 10 min at room temperature in a dark place. The cells were washed with 1% acetic acid three times and left overnight to air-dry. The protein-bound SRB stain was dissolved by adding 150 μL of 10 mM Tris base (pH 7.4). The O.D. was measured at 540 nm using the microplate reader FluoStar Omega (BMG, Labtec, Ortenberg, Germany). The dose–response curves of drugs under investigation were analyzed using the Emax model.23

2.6. Data Analysis

The dose–response curves of compounds were investigated by applying the Emax model as previously described.24 The combination index (CI) was calculated as previously described.25 Drug interactions are classified as an additive if CI is between 0.8 and 1.2, antagonistic if CI is > 1.2, and synergistic if CI is < 0.8.

2.7. Cell Cycle Analysis

DOX-resistant breast cancer cells (MCF-7ADR) were treated with the precalculated IC50 of hypophyllanthin (PN4) and phyllanthin (PN5) alone or in combination with DOX for 48 h. Then, the cells were harvested by trypsinization, twice washed with PBS (phosphate-buffered saline), fixed in ice-cold 60% ethanol at 40 °C, and re-washed in PBS. After that, the cells are resuspended in 500 μL of propidium iodide (PI) with RNase staining buffer, BD (Franklin Lakes, NJ, USA), and incubated for 30 min. Last, FACS analyses were executed utilizing the ACEA Novocyte flow cytometer (ACEA Biosciences Inc., San Diego, USA). For every sample, data from 12,000 cells were collected, and the distribution of cell cycle phases was analyzed using the ACEA Novo Express software (ACEA Biosciences Inc., San Diego, USA).26

2.8. Apoptosis Analysis

MCF-7ADR cells were treated with hypophyllanthin (PN4) and phyllanthin (PN5) alone or in combination with DOX for 48 h and then trypsinized and washed twice with PBS. Apoptosis assessment was performed via the Annexin V-FITC/PI Apoptosis Detection Kit (BD Biosciences, San Diego, USA) following manufacturer’s instructions. In brief, the cells were resuspended in 0.5 mL of the binding buffer, and then 5 μL of Annexin V-FITC and 5 μL of PI (staining solution) were added for 15 min at room temperature in a dark place. Finally, the cells were applied, within 1 h of staining, to FACS analysis using an ACEA Novocyte flow cytometer (ACEA Biosciences Inc., San Diego, CA, USA).27

2.9. Autophagy Assessment

For autophagic assessment in response to hypophyllanthin (PN4) and phyllanthin (PN5) alone or in combination with DOX treatment for 48 h, MCF-7ADR cells were trypsinized and washed twice with ice-cold PBS. Then, 0.5 mL of the staining solution (1 μg/mL of acridine orange in PBS) was added and incubated in the dark for 30 min at room temperature. The cells were adjusted at 12,000 events when applied to flow cytometric analysis via the ACEA Novocyte flow cytometer, and fluorescent signals were analyzed via a FL1 signal detector (488 nm excitation/530 nm emission). The net fluorescence intensities (NFI) were quantified.28

2.10. Cell Scratch Assay

MCF-7ADR cells were seeded at 90% confluency in 12-well plates in triplicate for each condition. After 24 h, a scratch was introduced across the center of each well using a 1 mm pipette tip. The cells were washed twice with PBS to remove cell debris and replaced with fresh media alone or in combination with PN4, PN5, and/or DOX. The cells migrated into the wound surface, and the average distance of migrating cells was determined by inverted microscopy at designated time points (0, 24, 48, 72, and 96 h).29

2.11. Western Blot Analysis

Cultured cells were harvested and lysed in a readymade RIPA buffer (Beyotime Biotechnology) containing a protease inhibitor cocktail (1 μg/mL aprotinin and leupeptin) to extract proteins from the cells. Proteins in the cell lysate (30 μg) were separated through SDS-PAGE gels and transferred to nitrocellulose membranes (Amersham Pharmacia Biotech, Buckinghamshire, UK). Membranes were blocked with 5% skimmed milk for 3 h; then, the membrane was incubated with the primary antibodies at 4 °C overnight. The membrane was washed with TBS-T buffer and then incubated with the corresponding secondary antibodies at 37 °C for 2 h. Protein signals were analyzed with a luminescent image analyzer (LAS-1000; Fujifilm, Tokyo, Japan) using the Immobilon Western Chemiluminescent HRP Substrate (Millipore, Burlington, MA, USA).30

3. Results

From our previous study,21 only the CH2Cl2 fraction at 100 μg/mL exerted cytotoxic activity against both MCF-7 and MCF-7ADR cell lines, and it was relatively more cytotoxic against MCF-7ADR. In addition, the CH2Cl2 fraction at 10 and 100 μg/mL improved the cytotoxic effect of DOX against both cell lines. Fractionation of the CH2Cl2 fraction using preparative TLC afforded four major fractions: B1, B2, B3, and B4. Among them, only the B2 fraction (at 100 μg/mL) was cytotoxic against MCF-7ADR but not MCF-7. In addition, fraction B2 at a concentration of 10 μg/mL showed an additive effect when combined with DOX. Therefore, the separation of the major components in the range of Rf of B2 fraction was carried out.

3.1. Isolation of Major Compounds from Bioactive Fraction

Column chromatography of the CH2Cl2 fraction afforded five compounds. The identity of the isolated compounds was confirmed by comparing their NMR data with those reported in the literature and by comparison to authentic samples. Spectral analysis of the isolated compounds revealed the identification of sterol (PN1) and four lignans (PN2–PN5) (Figure 1).

Figure 1.

Figure 1

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Compound s Isolated from P. niruri.

Compound PN1 was identified as β-sitosterol,22 by comparison with the standard sample (Co-TLC) and 1H NMR spectrum (Figure S1).

The NMR spectra (1H, 13C NMR, MS, and UV) of compounds PN2 to PN5 (Figures S2–S17 and Table S1) showed the common feature of aryl lignans. The result of NMR analysis revealed that compounds PN2 and PN4 are belonging to the aryltetralin skeleton based on the presence of only one benzylic CH2 group, while the presence of two benzylic CH2 groups in PN3 and PN5 indicated a dibenzylbutane skeleton.31,32 In addition, the spectral data of compounds PN2, PN3, and PN4 showed the presence of a methylene dioxy group (δH 5.80–5.89 and δC 100.0–101.0) and five methoxy groups, while compound PN5 showed the absence of the methylene dioxy group and the presence of six methoxy groups. From the previous discussion and by comparison of the spectral data (1H, 13C NMR, MS, and UV) of the isolated compounds with those reported in the literature, PN2 was identified as nirtetralin31,32 (Figures S2–S5), PN3 as niranthin31−33 (Figures S6–S9), PN4 as hypophyllanthin32−34 (Figures S10–S13), and PN5 as phyllanthin32−34 (Figures S14–S17).

3.2. Effect of the Isolated Compounds PN2–5 on the Proliferation of MCF-7 and MCF-7ADR

MCF-7 and MCF-7ADR cell lines were separately treated with the isolated compounds PN2, PN3, PN4, and PN5 over a concentration range of 0.01–100 μM for 72 h, and the viability was evaluated by the SRB assay (Figure 2A,B). PN2 and PN3 did not induce apparent cytotoxicity against both cell lines with IC50 values higher than 100 μM. Hypophyllanthin (PN4) and phyllanthin (PN5) exerted weak cytotoxic activity against MCF-7 cells with nearly the same IC50 values (74.2 ± 1.5 and 73.4 ± 2.1 μM, respectively). However, more potent cytotoxicity was observed against MCF-7ADR cells after treatment with PN4 and PN5 (with IC50’s values of 58.7 ± 1.2 and 29.5 ± 0.9 μM, respectively). Therefore, we further studied the influence of PN4 and PN5 on the efficacy profile of DOX in breast cancer-resistant cells MCF-7ADR, where PN5 was found to be 2-fold more potent than PN4.

Figure 2.

Figure 2

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Cytotoxic activity of the isolated compounds on MCF-7 and MCF-7ADR cells. MCF-7 (A) and MCF-7ADR (B) cells were treated with serial dilution of PN2–PN5 for 72 h. Cell viability was evaluated by the SRB assay, and data are expressed as mean ± SD (n = 3).

3.3. Effect of Hypophyllanthin (PN4) and Phyllanthin (PN5) on the Cytotoxic Profile of DOX in MCF-7ADR

To investigate the influence of PN4 and PN5 on the cytotoxic profile of DOX, we evaluated the dose–response curve of DOX alone relative to its effect when combined with PN4 and PN5 in MCF-7ADR cells (Table 1 and Figure 3). DOX alone exerted gradient cytotoxicity with increasing concentration with an IC50 value of 17.0 ± 1.9 μM. However, an equitoxic combination of PN4 or PN5 with DOX markedly improved the cytotoxic profile of DOX, as indicated by the decrease in its IC50 value to 9.5 ± 0.6 and 1.1 ± 0.03 μM, respectively. The determined combination index values for DOX with PN4 or PN5 were 0.65 and 0.091, respectively. This observation indicates a potent synergistic interaction, especially with PN5 in MCF-7ADR (Table 1).

Table 1. Comparative Cytotoxicity of DOX alone and in Combination with Hypophyllanthin (PN4) and Phyllanthin (PN5) against MCF-7ADRa.

  MCF-7ADR
treatments IC50 (μM) R-value (%) CI-value
DOX (alone) 17.0 ± 1.9 1.2 ± 0.05  
DOX + PN4 9.5 ± 0.6 5.4 ± 0.4 0.652 [synergism]
DOX + PN5 1.1 ± 0.03 6.2 ± 0.31 0.091 [synergism]
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a

The value of the IC50’s of DOX is determined alone and from the equitoxic ratios of DOX/PN4 (1:10) and DOX/PN5 (1:5). Data are presented as mean ± SD; n = 3.

Figure 3.

Figure 3

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Chemomodulatory effect of PN4 and PN5 on the cytotoxicity of DOX in the MCF-7ADR cell line. Cells were treated with serial dilution of DOX alone or in combination with PN4 and PN5 in equitoxic combination (1:10) and (1:5), respectively, for 72 h. Cell viability was evaluated by the SRB assay, and data are expressed as mean ± SD (n = 3).

3.4. Effect of Hypophyllanthin and Phyllanthin alone or in Combination with DOX on the Cell Cycle in MCF-7ADR Cells

DNA flow cytometry was used to assess the effect of hypophyllanthin and phyllanthin alone or in combination with DOX on the cell cycle distribution of the MCF-7ADR cell line (Figure 4). PN4 showed no significant change in the cell population of the G0/G1-phase (the non-proliferating cell fraction) and the S phase compared to the control cells, but PN4 significantly decreased the cell population in the G2/M phase from 22.19 ± 1.2 to 18.37 ± 0.95% concomitantly with doubling cell population in the pre-G phase from 1.46 ± 0.01 to 2.91 ± 0.03%.

Figure 4.

Figure 4

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(A) MCF-7ADR cells were exposed to DOX, (hypophyllanthin) PN4, (phyllanthin) PN5, and their combinations for 48 h, and cell cycle distribution was determined using DNA cytometry analysis. (B) Cell phase percentages were plotted as the percent of total events. (C) Pre sub-G phase was plotted as the percentage of total events. Data are presented as mean ± SD; n = 3.

Unlikely, phyllanthin (PN5) treatment showed significant changes in all cell phases compared to control cells. PN5 caused a meaningful increase in the Go/G1 phase from 54.13 ± 1.3 to 64.40 ± 1.6% that resulted in cell death manifested by a significant increase in the pre-G phase from 1.46 ± 0.01 to 7.32 ± 0.2% compared to control cells. Reciprocally, PN5 caused a substantial decrease in the S phase from 23.69 ± 1.7 to 16.67 ± 0.9% and in the G2/M phase from 22.19 ± 1.2 to 18.92 ± 0.77%.

Compared to control cells, treatment with DOX resulted in a significant cell cycle arrest at the G2/M phase from 21.8 ± 1.6 to 75.87 ± 2.14%, indicating the difficulty of repairing the intracellular damaged DNA. Moreover, DOX treatment resulted in a significant cell death manifested by a marked increase in the Pre-G phase from 1.46 ± 0.01 to 30.17 ± 1.8% compared with control cells.

The combination of DOX with PN4 caused a very potent antiproliferative effect compared to DOX treatment alone as manifested by the increase in the cell population in the G0/G1 phase from 6.34 ± 0.2 to 36.13 ± 2.53% and in the S phase population from 17.79 ± 1.06 to 32.88 ± 1.8%, while this combination caused a significant decrease in the Pre-G phase cell population from 30.17 ± 1.8 to 18.37 ± 0.56% compared to DOX treatment alone.

Unlikely, the combination of PN5 with DOX significantly induced more cell death when compared to DOX treatment alone; cells in the pre-G phase were increased from 30.17 ± 1.8 to 55.75 ± 2.78%. In addition, the combination of PN5 with DOX also showed antiproliferative effect and caused a significant increase in the G0/G1 phase from 6.34 ± 0.12 to 47.21 ± 0.66% and in the S phase from 17.79 ± 1.06 to 22.44 ± 1.2% when compared to DOX alone.

3.5. Modes of Cell Death Involved in the Synergistic Effect of Combined Treatment

To discern if hypophyllanthin (PN4) and phyllanthin (PN5) induced their cytotoxic activity via apoptosis or necrosis, MCF-7ADR cells were treated with the IC50 values of PN4 and PN5 alone and in combination with DOX for 48 h, and then the cells were assessed using PI and Annexin-V/FITC staining coupled with flow cytometry. In MCF-7ADR cells, treatment with PN4 and PN5 showed no significant apoptotic or necrotic cell death compared to control cells (Figure 5A,B).

Figure 5.

Figure 5

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(A) Apoptosis/necrosis assessment in MCF-7ADR cells after treatment with DOX, (hypophyllanthin) PN4, (phyllanthin) PN5, and their combinations for 48 h. Cells were stained with PI/Annexin V-FITC. (B) Different cell populations were plotted as the percentage of total events. Data are presented as mean ± SD; n = 3.

DOX is a chemotherapeutic drug that can damage DNA and alter biological processes, such as apoptosis and necrosis. Herein, DOX alone caused a significant increase in total apoptosis (early and late) from 1.1 ± 0.17 to 24.2 ± 1.9% and necrosis from 0.62 ± 0.06 to 59.9 ± 1.13% compared to control untreated MCF-7ADR cells (Figure 5B).

Compared to DOX alone, the combination of DOX with PN4 and PN5 resulted in more total cell death. DOX combined with PN4 induced further significant necrosis (79.91 ± 0.15 vs 60.4 ± 1.13% of DOX alone), while the combination of DOX with PN5 induced further significant apoptosis (35.4 ± 1.35 vs 24.7 ± 1.9% of DOX alone). These data provided more about different modes of cell death elicited by PN4 and PN5 alone and as an adjuvant to DOX against MCF-7ADR cells.

3.6. Reversal of ADR Resistance by Hypophyllanthin and Phyllanthin in Combination with DOX Is Linked to Autophagy Inhibition

Currently, the autophagy role in cancer resistance to DOX and the involved mechanisms have become an area of interest to many investigators. Hence, to verify whether the reversal of ADR resistance by hypophyllanthin (PN4) or phyllanthin (PN5) in combination with DOX is related to autophagy, MCF-7ADR cells were separately treated with PN4 and PN5 alone and in combination with DOX for 48 h, and autophagy was assessed using acridine orange dye coupled with flow cytometry (Figure 6A).

Figure 6.

Figure 6

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(A) Autophagic cell death evaluation in MCF-7ADR cells after exposure to DOX, hypophyllanthin (PN4), and phyllanthin (PN5) alone or in combination with DOX for 48 h. The cells were stained with acridine orang dye. (B) NFI was plotted in comparison with the basal fluorescence of untreated MCF-7ADR cells. (C) Western blot to detect the expression of the autophagy-related protein (LC3II). Data are displayed in triplicate.

The results showed that DOX treatment alone significantly induced autophagic signal by nearly 100% (2-fold increase) compared to untreated control cells, further indicating that autophagy is associated with DOX resistance. However, the treatment of hypophyllanthin and phyllanthin combined with DOX markedly attenuated the autophagic induction of DOX by 37 and 40%, respectively (Figure 6B), suggesting that PN4 and PN5 combinations may reverse DOX autophagy-related resistance in MCF-7ADR. Next, we further detected the autophagy-associated protein LC3II/I by western blotting. As shown in Figure 6C, DOX markedly enhanced the expression level of LC3II/I compared to that of control cells, while PN4 or PN5 combinations inhibited its expression compared to DOX alone.

On the other hand, treatment with PN4 alone significantly induced the autophagic signal higher than control cells by 54%, while treatment with PN5 inhibited autophagy by 15% compared with control cells (Figure 6B), indicating opposing effects of each compound alone in autophagy. Indeed, the results showed that compared with the control, PN4 alone enhanced the expression of autophagy-associated protein LC3II/I, while a slight reduction in LC3II/I expression was observed after treatment with PN5 (Figure 6C).

3.7. Hypophyllanthin and Phyllanthin Overcome Resistance of DOX in MC F-7ADR through Downregulating SIRT1/Akt

Research suggested a critical role of the SIRT1/Akt axis in the progression of breast cancer. SIRT1 was found to maintain the genomic integrity that regulates DOX resistance in breast cancer by activating the Akt pathway. To examine whether the inhibitory effect of hypophyllanthin (PN4) and phyllanthin (PN5) on MCF-7ADR cells was associated with SIRT1 and Akt, MCF-7ADR cells were untreated or treated with DOX or in combination with PN4 or PN5 for 48 h, and expressions of SIRT1 and P-Akt were assessed by western blot (Figure 7).

Figure 7.

Figure 7

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Hypophyllanthin (PN4) and phyllanthin (PN5) enhanced the effect of DOX by reducing the SIRT1/Akt pathway in MCF-7ADR . The cells were treated with PN4 and PN5 alone and in combination with DOX for 48 h; then, cells were collected to detect the protein expressions of SIRT1 and P-Akt by western blot analysis. Data are presented as mean ± SD.

Cells treated with DOX alone exhibited a decrease in the levels of SIRT1 and P-Akt, whereas treatment with PN4 and PN5 was more potent than DOX alone (compared with untreated control cells) in decreasing the levels of SIRT1 and P-Akt. Also, treatment with DOX in combination with PN4 or PN5 significantly reduced the expression of SIRT1 and AKT compared to DOX alone. Hence, we hypothesize that PN4 and PN5 can ameliorate DOX resistance in a SIRT1-dependent manner via decreased SIRT1 and P-Akt expressions in MCF-7ADR cells.

3.8. Hypophyllanthin and Phyllanthin Inhibited the Migration Ability of MCF-7ADR Cells and Downregulated the Expression of EMT Markers

Herein, we evaluated the effect of hypophyllanthin (PN4) and phyllanthin (PN5) on cell migration of MCF-7ADR via the cell scratch assay. Yet, cells were treated with PN4, PN5, and DOX either alone or in combination, and wound closure was assessed daily until the closure of control untreated cells (96 h).

The results of the cell scratch test suggested that the migratory potential of MCF-7ADR cells was not significantly affected after treatment with DOX alone compared to untreated cells, while both PN4 and PN5 showed a notable decrease in cell migration of MCF-7ADR cells compared to untreated cells, indicating the potential antimigratory effect of both compounds.

Interestingly, the combination treatment of DOX with PN4 and PN5 achieved a remarkably more potent migration-inhibitory effect than DOX (Figure 8A,B), giving more evidence about their potential effect in inhibiting MCF-7ADR migration and enhancing the DOX effect in controlling the migration of these resistant cells.

Figure 8.

Figure 8

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Hypophyllanthin (PN4) and phyllanthin (PN5) alone and in combination with DOX inhibit cell migration of MCF-7ADR cells. (A) The migration distances were measured after different treatments daily for 96 h. (B) Data were blotted as wound closure % at each time interval. (C) The protein level of EMT-related proteins (β-catenin and N-cadherin) was determined by western blot analysis in MCF-7ADR cells. Data are presented as triplicates.

Recent research suggests the involvement of EMT in the sensitivity of breast cancer cells to DOX treatment. Since MCF-7ADR cells exhibited EMT-like phenotype and enhanced metastatic potential, thus we tested the expressions of EMT-related proteins, specifically mesenchymal cell markers (N-cadherin and β-catenin) by western blot analysis after treatment with PN4 and PN5 and/or DOX (Figure 8C). We observed that the protein expression level of N-cadherin decreases after treatment with DOX, which indicates that DOX administration to MCF-7ADR cells might suppress EMT. Additionally, it was observed that the protein level of N-cadherin and β-catenin decreased significantly after treatment with predetermined IC50 of PN4 and PN5 compared with the untreated or DOX-treated cells. This observation indicates that exposure of MCF-7ADR cells to PN4 or PN5 may further suppress EMT. Also, in the cells treated with a combination of DOX with PN4 or PN5, the expression of N-cadherin and β-catenin proteins was downregulated compared with DOX alone. These results indicate that PN4 and PN5 can enhance the MCF-7ADR cells’ sensitivity to DOX by EMT inhibition.

4. Discussion

Despite the remarkably improved prognosis of breast cancer patients diagnosed at early stage and received standard chemotherapy after surgical resection, the 5 year survival rates of breast cancer patients remain high, mainly due to drug resistance.35 Moreover, for patients with advanced and metastatic cancers who have no opportunities to operate, chemotherapy is considered the most efficient approach to alleviate symptoms and improve survival rate. Yet, a subset of patients presents poor survival outcomes owing to drug resistance.36 Therefore, overcoming resistance is still a big challenge in breast cancer chemotherapy.37

DOX is a commonly used chemotherapeutic drug in breast cancer which is affected by the development of acquired drug resistance and EMT upon prolonged use, resulting in chemotherapy failure.38 Accordingly, we are investigating several chemomodulators to work as a sensitizing agent to DOX particularly in resistant breast cancer cells.39,40

Herein, we showed for the first time that two lignans, hypophyllanthin and phyllanthin, separated from P. niruri, significantly inhibited the growth and migration capacity of DOX-resistant breast cancer cells (MCF7ADR cells) when combined with DOX.

Surprisingly, the killing effect of compounds isolated from P. niruri (PN2–PN5) was more potent in MCF-7ADR cells compared to the naïve MCF-7 cells. Moreover, MCF-7ADR cells became more susceptible to DOX in the presence of hypophyllanthin and phyllanthin, indicating a sensitization effect of both compounds in resistant breast cancer cells. Accordingly, we further investigated the potential underlying mechanisms of this sensitization in MCF-7ADR cells.

Phyllanthus species have been studied in recent decades for cancer treatment. However, few studies were concerned with P. niruri constituents in various plant parts and their biological activities, especially in cancer.41 In general, studies investigating the anticancer properties of Phyllanthus species on breast cancer were scanty. Earlier in 2014, a study examined the in vitro and in vivo anticancer potential of hypophyllanthin and phyllanthin isolated from P. amarus against breast cancer.42 Another study showed that four Phyllanthus species inhibit metastasis of human breast cancer cells via the suppression of ERK1/2.43 Recently in 2021, a study showed the effective combination of P. niruri extract with Curcuma longa extract for treating metastatic breast cancer stem cells.18 Herein, this is the first study investigating the potential anticancer activity of compounds derived from Phyllanthus species against resistant breast cancer cells.

Several studies establish that the development, metastasis, and recurrence of tumors are closely correlated with the cell cycle.44,45 In addition, one of the strategies for improving the sensitivity of cancer cells to chemotherapeutics is to combine them with cell cycle regulators.46 Hypophyllanthin alone caused a significant decrease in the G2/M phase, while phyllanthin alone caused a significant increase in the G0/G1 phase. This indicates the antiproliferative potential of hypophyllanthin and phyllanthin, and they can hinder the progression of the cell cycle in MCF-7ADR cells. Our study revealed a prominent G2/M phase arrest for MCF-7ADR cells after DOX treatment, consistent with several previous studies.47,48 This cell cycle arrest-induced stress ultimately induces apoptosis in breast cancer.40 On the contrary, DOX-treated MCF-7ADR cells after co-treatment with hypophyllanthin and phyllanthin were markedly accumulated in the G0/G1 phase, suggesting that hypophyllanthin and phyllanthin distinctly modulated cell cycle arrest induced by DOX and revealed the potential of hypophyllanthin and phyllanthin as co-chemotherapeutic agents. Moreover, causing the G0/G1 phase arrest of MCF-7ADR after co-treatment indicated the potent antiproliferative effects of both combinations. Phyllanthin exerted a more potent antiproliferative activity than hypophyllanthin that was accompanied by more cell death and more cells undergoing DNA fragmentation represented by the increase in pre-G population, compared with DOX alone. On the other hand, co-treatment with hypophyllanthin showed less cell death represented by a decrease in pre-G population.

In conclusion, the combination with hypophyllanthin was more cytostatic than cytotoxic. These results encouraged us to continue biological investigations on such combinations to understand the targeted mechanism of combination treatments by assessing apoptotic and autophagy-programmed cell death.

Among its other cellular effects, DOX induces ER stress and dysregulate autophagy, and on top of inducing apoptosis, DOX might induce necrotic cell death.49,50 Consistent with these previous studies, our data revealed that DOX effectively induced cell apoptosis as well as necrosis in MCF7ADR cells. Studies that investigated apoptosis induction of Phyllanthus nuri on cancer cells were scanty. Our study revealed that each of hypophyllanthin and phyllanthin alone did not induce significant apoptosis in MCF-7ADR. However, the combination of hypophyllanthin with DOX shifted cell death more toward necrosis, while phyllanthin combination with DOX showed an exceptional synergistic effect in terms of apoptosis, suggesting that phyllanthin cytotoxicity is mainly via apoptosis induction. Apoptosis is the preferred mechanism of cancer cell death in response to chemotherapy because necrosis induces inflammatory collateral effects which are not desirable.51 Therefore, apoptosis is considered an ideal target in cancer therapy, and consequently, phyllanthin combination with limited necrosis is more promising in resistant breast cancer cells.

Despite the complex and controversial role of autophagy in cancer, either as a pro-survival or pro-death, studies demonstrated autophagy as a new reversal strategy in multidrug resistance cancer therapy. Recently, autophagy-mediated drug resistance in tumor cells has been widely investigated,6 with high autophagy levels noted in resistant breast cancer cells. DOX simultaneously triggers differently regulated cell death pathways, including induction of autophagy, at low doses.39 In line with these studies, we found that DOX treatment activates autophagy in MCF7ADR cells, confirmed with the high expression of LC3II (autophagy golden marker), and providing stronger evidence that the autophagy is associated herein with DOX resistance.52,53 More and more preclinical data are concerned with reversing DOX resistance through modulating autophagy as one of the promising therapeutic strategies.54 In this context, autophagy acts as a salvage mechanism or apoptosis-escape mechanism advocative for drug resistance. Various Phyllanthus species inhibited autophagy in several cancers like gastric cancer,55 ovarian cancer,56 and HCC.57 Herein, we reported for the first time the effect of hypophyllanthin and phyllanthin on autophagy in resistant breast cancer cells. In this study, we showed that hypophyllanthin and phyllanthin combined with DOX inhibited autophagy by inhibiting the expression of LC3II, thereby reversing drug resistance. However, treatment with hypophyllanthin and phyllanthin alone showed opposing mechanisms regarding autophagy. Hypophyllanthin activated autophagy, while phyllanthin inhibited autophagy, confirming the different modes of cell death induced by the two compounds and explaining in part the observed potent sensitizing effect of phyllanthin over hypophyllanthin.

To date, more and more strategies to overcome resistance are concerned with the roles of SIRT1 in breast cancer progression and the development of treatment resistance.9 SIRT1, a member of the mammalian sirtuin protein family, is significantly involved in several biological processes, including DNA repair, gene silencing, cell survival, metabolism, and aging.58 Previous studies showed discrepancies in the expression of SIRT1 within different tumors, suggesting that SIRT1 can function either as a promoter or a suppressor in chemotherapy resistance. In breast cancer, SIRT1 expression was upregulated in drug-resistant cancer cell lines through increasing expression level of multidrug-resistant protein 1, suggesting SIRT1’s promotional role in chemoresistance. Previous research revealed the crosslink between SIRT1 and Akt in the progression of breast cancer,59 suggesting that SIRT1 directly activates Akt. In breast cancer, overactivation of Akt can cause uncontrolled cell proliferation and prevent programmed cell apoptosis. Consequently, disrupting the interrelation between SIRT1 and Akt would lead to inhibiting proliferation and invasive ability of MCF-7ADR cells. In the current study and as expected, DOX modulates the expression of SIRT1 in MCF-7ADR cells, which might be influencing DOX resistance. Currently, non-specific SIRT1 inhibitors like tenovin-6 and cambinol suppress tumor growth and can be utilized in different therapeutic purposes.60 Herein, we found that hypophyllanthin and phyllanthin treatment alone or in combination with DOX are significantly downregulating the expression of SIRT1 and its downstream activity marker, p-Akt protein, in MCF7ADR cells, providing a new insight into the potential use of hypophyllanthin and phyllanthin in the treatment of resistant breast cancer phenotype. In addition, SIRT1 signaling is reported to be associated with EMT transcriptional factors in cancer, which in turn enhances cancer cell migration, invasion, and metastasis.61,62 EMT is a biological transformation of epithelial cells to mesenchymal cells through which cellular polarity and connections are lost, which leads to stronger migratory and invasive cancer cell properties. EMT not only promotes metastasis of cancer cells but also enhances the development of DOX resistance.63 EMT transcriptional factors are overexpressed in MCF-7ADR cells, and such resistant tumors carry more mesenchymal properties and are 3 times less likely to respond to chemotherapy than non-resistant cells.

Hence, the identification of novel agents which can inhibit EMT is of ultimate interest in improving the response of resistant tumors. Resistance to DOX treatment was also reported to be mediated by EMT in cancers via complex mechanisms, among which the β-catenin signaling pathway is the most frequently reported pathway.64,65 In the present study, β-catenin was highly expressed in MCF7ADR cells, confirming its essential role in DOX resistance of breast cancer.66 Interestingly, it was indicated for the first time that hypophyllanthin and phyllanthin decreased the migration capacity of DOX-resistant breast cancer cells (MCF-7ADR) and suppressed the expression of EMT markers (N-cadherin) and EMT inducer (β-catenin) together with synergizing the activity of DOX. These data suggested that hypophyllanthin and phyllanthin had an inhibitory effect on the EMT phenotype of MCF7ADR cells.

5. Conclusions

Hypophyllanthin and phyllanthin isolated from P. niruri showed a moderate antiproliferative/cytotoxic property against resistant breast cancer cells. Yet, both agents significantly synergized DOX-induced anticancer properties against resistant breast cancer cells. This enhanced activity was explained by further promoting DOX-induced apoptosis and suppressing the apoptosis-escape autophagy feature of the resistant breast cancer cells. On the molecular level, both compounds (hypophyllanthin and phyllanthin) interfere with the SIRT1/Akt pathway and suppress N-cadherin/β-catenin axis, confirming the observed antiproliferative, cytotoxic, and anti-invasive effects of hypophyllanthin and phyllanthin.

Acknowledgments

The authors would like to thank the Natural Wellness Biotech Co. and Ministry of Agriculture Malaysia initiative for funding the current research under the New Key Economic Areas Entry Point Project on High Value Herbals. This study was also partially funded by Prince Sattam bin Abdulaziz University (project number: PSAU/2023/R/1444).

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.3c02953.

  • ESI-MS, 1H, and 13C NMR data for compounds PN1–5 (PDF)

Author Contributions

M.R.M., A.E.H, A.M.A.-A, and E.A.-S. contributed to conceptualization; O.E.A.S., R.M.A., S.H.S., M.K., and A.E.H. contributed to methodology; O.E.A.-S., R.M.A., S.O.M., A.M.E.-D., and A.E.H. contributed to investigation; all authors contributed to writing—original draft preparation; all authors contributed to writing—review and editing; M.R.M. and A.M.A.-A contributed to supervision; and M.R.M., A.M.A.-A, A.E.H, S.H.S., M.K., and E.A.-S. contributed to project administration. All authors have read and approved the final manuscript.

The current research was funded by the Ministry of Agriculture Malaysia initiative under Point Project on High Value Herbals winned by Natural Wellness Biotech.

The authors declare no competing financial interest.

Supplementary Material

ao3c02953_si_001.pdf (1.3MB, pdf)

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