Skip to main content
NCBI home page
BMC Cancer logoLink to BMC Cancer
. 2025 Jul 1;25:1023. doi: 10.1186/s12885-025-14276-9

Excavatolide C has oxidative-stress-dependent antiproliferative and apoptotic effects against breast cancer cells

Jun-Ping Shiau 1,2,#, Che-Wei Yang 3,#, Wangta Liu 4, Szu-Yin Yu 3, Chia-Hung Yen 3, Fang-Rong Chang 3, Jyh-Horng Sheu 5,6,✉, Hsueh-Wei Chang 7,8,9,✉
PMCID: PMC12211348  PMID: 40596939

Abstract

Background

Triple negative breast cancer (TNBC) shows a poor response to targeted therapy drugs for non-triple-negative breast cancer (non-TNBC). Developing anticancer drugs that are effective for both TNBC and non-TNBC cells is necessary. The marine coral Briareum excavatum-derived excavatolide C (EXCC) exhibits anti-bladder cancer cell proliferation. However, the anti-breast cancer properties and drug safety of are unclear.

Methods

This study aimed to evaluate the antiproliferative effect and mechanisms (oxidative stress, DNA damage, and apoptosis) caused by EXCC on TNBC and non-TNBC cells in parallel with normal cells.

Results

EXCC demonstrated higher antiproliferative effects in various breast cancer cell lines (MDA-MB-231, Hs578t, MDA-MB-468, and MCF7) than in normal breast cell lines (H184B5F5/M10; M10) as detected in a 48 h ATP assay. MDA-MB-231 and MCF7 were chosen as representative TNBC and non-TNBC cells, respectively, to clarify the underlying molecular mechanisms. EXCC highly upregulated reactive oxygen species and mitochondrial superoxide, reduced the mitochondrial membrane potential, and downregulated glutathione in breast cancer compared with normal cells. These EXCC-triggered antiproliferative and oxidative stress changes were attenuated by the ROS inhibitor N-acetylcysteine (NAC). Consistently, in breast cancer cells, EXCC triggered subG1 accumulation, apoptosis, caspase activation, and DNA damage (γH2AX and 8-hydroxy-2’-deoxyguanosine), all of which were alleviated by NAC.

Conclusion

Overall, the antiproliferative effects and molecular mechanisms caused by EXCC in breast cancer treatment depend on oxidative stress. Without cytotoxicity to normal cells, EXCC is a potential antiproliferative marine natural product for TNBC and non-TNBC cells.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12885-025-14276-9.

Keywords: Soft coral, Diterpenes, Breast cancer, Oxidative stress, Apoptosis, DNA damage

Introduction

Breast cancer was one of the leading causes of women’s deaths from cancer according to the 2024 United States Cancer Statistics [1]. The estimated numbers of new cases and deaths were 310,720 and 42,250 for female breast cancer patients. The typical therapy for breast cancer is surgery with adjuvant chemotherapy [2]. However, chemotherapy may have adverse effects on breast cancer patients [3].

Moreover, breast cancer is broadly classified as triple-negative breast cancer (TNBC) and non-TNBC based on the expression status of hormone receptors and human epidermal growth factor 2 receptor [4]. TNBC, which accounts for 10 to 15% of breast cancers, is generally more aggressive, is more likely to recur, and has worse survival than non-TNBC [5]. Targeted therapy focusing on estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2), such as Fulvestrant [6], Everolimus [7], and Trastuzumab [8], has been developed to treat non-TNBC patients but is unsuitable for TNBC patients. This warrants the development of drugs that are functional for both TNBC and non-TNBC and do not cause damage to normal cells.

Marine coral is a rich source of natural products [9, 10] that exhibit anticancer effects [11–14]. Several Briareum excavatum-derived compounds, such as excavatolide B to M, have been identified [15, 16]. Many reports in the literature have identified diverse functions of excavatolide B, such as anti-inflammatory [17], anti-lung cancer [18], anti-osteoclastogenesis [19], and anti-angiogenesis [20] effects.

In comparison, excavatolide C (EXCC) was identified in 1998 [15], but its biofunctions have been only sporadically reported. The potential anticancer effects of EXCC have only been reported for bladder cancer cells [21]. The impact of EXCC on the proliferation of other cancers has not been investigated, particularly for breast cancer cells. EXCC’s drug safety in normal cells has also not been assessed.

This study evaluated the antiproliferative potential of EXCC against TNBC and non-TNBC cells and its safety for normal cells. The mechanisms underlying the role of oxidative stress in modulating DNA damage and apoptotic cell death were explored.

Materials and methods

EXCC, oxidative stress scavenger, and apoptosis inhibitors

The Gorgonian B. excavatum-derived EXCC was isolated and purified as described in our previous work [15, 22]. Its purity (> 95%) was examined using NMR spectroscopy [21].

N-acetylcysteine (NAC; 10 mM) (Sigma–Aldrich, St. Louis, MO, USA) [23–25] and Z-VAD-FMK (ZVAD; 50 µM) were chosen to inhibit oxidative stress and apoptosis through pretreatment for 1 and 2 h before adding EXCC for 48 h, namely, NAC/EXCC and ZVAD/EXCC.

Cells, viability, and selectivity index

Four breast cancer cell lines (MCF7, MDA-MB-231, Hs578t, and MDA-MB-468) were selected from ATCC (Manassas, VA, USA). H184B5F5/M10 (M10), the nonmalignant breast epithelial cell line, was purchased from BCRC (Hsinchu, Taiwan). These breast cancer and M10 cells were cultured in DMEM/F12 medium (3:2 mixture) and alpha medium, mixed with antibiotics and 10% bovine serum or 10% fetal bovine serum (Gibco, Grand Island, NY, USA). The seeding numbers/wells in 96-well plates were 3,500 (MCF7 and MDA-MB-468), 3,000 (MDA-MB-231 and Hs578t), and 4,500 (M10). After overnight, cells were received with drug treatment. An ATP content detection kit (PerkinElmer Life Sciences, Boston, MA, USA) was chosen to determine the cell viability [22]. The selectivity index (SI) was calculated by the ratio of EXCC’s IC50 values to normal cells (M10)/breast cancer cells.

Cell cycle (subG1, G1, S, and G2/M)

The levels of cellular DNA stained with 7-aminoactinomycin D (Biotium, Inc., Hayward, CA, USA) (7AAD) were monitored for cell cycle determination. The seeding numbers/wells in 6-well plates were 150,000 for all cell types. After overnight, cells were received with drug treatment. The cells were pre-fixed overnight at 4oC with 75% ethanol and stained with 7AAD (1 µg/mL for 30 min) [26]. Flow cytometry analysis (Guava easyCyte, Luminex, TX, USA) was conducted after washing and resuspending the stained cells in PBS.

Apoptosis

Annexin V/7AAD is a common method for detecting apoptosis [27, 28]. The seeding numbers/wells in 12-well plates were 60,000 for all cell types. After overnight, cells were received with drug treatment. Cells were reacted with the Annexin V (1:1000)/7AAD (1 µg/ml) kit (Strong Biotech Inc., Taipei, Taiwan) for 30 min according to the instructions in the user manual. Flow cytometry analysis was conducted after washing and resuspending the stained cells in PBS.

Apoptotic signaling for caspases 3, 8, and 9 (flow cytometry)

The activation statuses of extrinsic, intrinsic, and terminal apoptotic proteins (caspases 3, 8, and 9) were analyzed using OncoImmunin kits (Gaithersburg, MD, USA) [29–31], i.e., PhiPhiLux-G1D2, CaspaLux8-L1D2, and CaspaLux9-M1D2. When these caspases are activated, their specific substrates are cleaved and generate fluorescence proportional to the degree of caspase activation. The seeding numbers/wells in 12-well plates were 60,000 for all cell types. After overnight, cells were received with drug treatment. Briefly, the cells were reacted with these caspase substrates (1:1000) at 37oC for 1 h. Flow cytometry analysis was conducted after washing and resuspending the stained cells in PBS.

Apoptotic signaling for caspases 3, 8, and 9 (Western blotting)

Caspases 3, 8, and 9 are terminal, extrinsic, and intrinsic signal proteins for apoptosis. Apoptotic signaling antibodies, such as cleaved caspase-3 (Asp175) (5A1E), cleaved caspase-8 (Asp374) (18C8), and cleaved caspase-9 (Asp330) (E5Z7N) rabbit mAb (#9664, #9496, and #52873) (Cell Signaling Technology, Danvers, MA, USA), were used to detect the activation (cleavage) of caspases 3, 8, and 9 via Western blotting as described previously [22]. 45 µg protein/lane was loaded and PVDF membranes were used in protein transfer for Western blotting. The blots were cut before hybridization with antibodies during blotting. After the first antibody was used to detect a target, some cut blots were stripped for the next target sequentially.

Reactive oxygen species (ROS) and glutathione (GSH)

GSH is a cellular antioxidant exhibiting free radical scavenging and antioxidation function [32]. The imbalance of ROS and GSH causes oxidative stress. Accordingly, ROS and GSH must be further assessed to observe oxidative stress. The cellular ROS and GSH levels were monitored with their reacting dyes, i.e., 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) (Invitrogen, Eugene, OR, USA) [33] and 5-chloromethylfluorescein diacetate (CMF-DA) [27] (Thermo Fisher Scientific, Carlsbad, CA, USA), respectively. The seeding numbers/wells in 12-well plates were 60,000 for all cell types. After overnight, cells were received with drug treatment. Briefly, the cells were reacted with 2 µM of DCFH-DA and 5 µM of CMF-DA for 30 min at 37oC. Flow cytometry analysis was conducted after washing and resuspending the stained cells in PBS.

Mitochondrial superoxide (MitoSOX) and mitochondrial membrane potential (MMP)

MitoSOX and MMP levels were monitored via their reacting dyes, i.e., MitoSOX™ Red [34] (Sigma–Aldrich) and DiOC2 (3) [27] (Invitrogen). The seeding numbers/wells in 12-well plates were 60,000 for all cell types. After overnight, cells were received with drug treatment. Briefly, cells were reacted with 5 µM of DiOC2 (3) and 50 nM of MitoSOX Red for 30 min at 37oC. Flow cytometry analysis was conducted after washing and resuspending the stained cells in PBS.

γH2AX and 8-hydroxy-2’-deoxyguanosine (8-OHdG)

γH2AX and 8-OHdG, markers for DNA double-strand breaks and oxidative DNA damage, were detected using an antibody recognition strategy [27]. The seeding numbers/wells in 6-well plates were 150,000 for all cell types. After overnight, cells were received with drug treatment. These assays need a fixation step before adding a primary antibody. Briefly, a γH2AX primary antibody (cat. no. SC-517348, Santa Cruz Biotechnology, Santa Cruz, CA, USA), a secondary antibody labeled with Alexa Fluor®488 (Cell Signaling Technology, Beverly, MA, USA), and 7AAD were subsequently incubated as described [27]. Meanwhile, the 8-OHdG antibody labeled with FITC (Santa Cruz Biotechnology) was incubated directly without a secondary antibody [27]. Flow cytometry analysis was conducted after washing and resuspending the stained cells in PBS.

Statistical analysis

The JMP12 statistical software developed by the SAS Institute (Cary, NC, USA) was applied to analyze the data with ANOVA, followed by Tukey’s HSD test. This software provides multiple comparisons by giving the different groups specific letters. No overlapping letters between treatments reveals significant changes (p < 0.05).

Results

EXCC shows antiproliferative effects

The antiproliferative effects of EXCC were evaluated in breast cancer (MDA-MB-468, MDA-MB-231, MCF7, and hs578t) and nonmalignant (M10) cells. EXCC demonstrated a more effective suppression of breast cancer cell viability than normal cells (M10) (Fig. 1A), i.e., IC50 = 26.07, 30.36, 33.8, 36.43, and 198.80 µg/ml, respectively. The selectivity indices (SI) for MDA-MB-468, MDA-MB-231, MCF7, and hs578t breast cancer cells were 7.62, 6.54, 5.88, and 5.45, regarding their IC50 values compared to nonmalignant cells (M10). Compared with clinical drugs, EXCC was slightly less suppressive than cisplatin on MCF7 and MDA-MB-231 cells under 48 h ATP assay. To address the role of ROS in EXCC-induced antiproliferative effects, the ROS inhibitor NAC was pretreated before EXCC. EXCC’s proliferation-inhibiting effects on breast cancer cells were attenuated by NAC (Fig. 1B). This result suggests that the antiproliferation of EXCC is mediated by ROS induction.

Fig. 1.

Fig. 1

Open in a new tab

Cell viability effects of EXCC. (A) Cell viability (ATP) assay for EXCC and cisplatin. M10 is a nonmalignant breast epithelial cell line; the others are breast cancer cell lines. Cells were treated with control (0.1% DMSO) and EXCC or cisplatin for 48 h. (B) Cell viability assay for NAC/EXCC. After NAC 1 h pretreatment, cells were exposed to EXCC for 48 h, i.e., NAC/EXCC. Data are shown as means ± SD (n = 3). In B, EXCC (black) 0, 25, and 50, indicating “a, b, and c” (non-overlapping), reveal significant outcomes for multi-comparisons. EXCC 0 (black) and NAC/EXCC (gray) 0, 25, and 50, indicating “a” (overlapping), reveal non-significant outcomes

Since the antiproliferative effects were similar in these cancer cells, we selected representative non-TNBC and TNBC cell lines, i.e., MCF7 and MDA-MB-231, for the following experiments.

EXCC shows cell-cycle-modulating effects

Regarding the flow cytometry assay, breast cancer cells (MCF7 and MDA-MB-231) were accumulated in subG1 (%) to a greater extent after EXCC treatment than were nonmalignant cells (M10), particularly at 50 µg/ml (Fig. 2). Moreover, EXCC’s subG1-accumulating effects on breast cancer cells were attenuated by NAC (Fig. 2).

Fig. 2.

Fig. 2

Open in a new tab

Cell cycle effects of EXCC and NAC/EXCC. Breast cancer (MCF7 and MDA-MB-231) and nonmalignant cells (M10) were incubated with EXCC for 48 h with or without NAC 1 h pretreatment, i.e., NAC/EXCC and EXCC. Non-overlapping letters reveal significant outcomes (p < 0.05). Data are expressed as means ± SD (n = 3)

In addition, MDA-MB-231 cells had a decreased G1 population at 25 and 50 µg/ml of EXCC and an increased G2/M population at 50 µg/ml (Fig. 2). MCF7 cells had an increased G1 population at 12.5 and 50 µg/ml of EXCC and an increased G2/M population at 25 µg/ml. In comparison, normal cells (M10) had an increased G1 population and a decreased G2/M population at 50 µg/ml of EXCC, while other treatments showed only mild changes. These results demonstrate that EXCC induced differential cell cycle disturbance and that breast cancer cells experienced more severe changes compared with normal cells. All of these cell cycle disturbances with EXCC were attenuated by NAC.

EXCC shows apoptosis-modulating effects

Annexin V (+) (%) was increased to a greater extent in breast cancer cells (MCF7 and MDA-MB-231) than nonmalignant cells (M10) treated with EXCC at various concentrations and for different times (Fig. 3A and B). Moreover, the Annexin V-increasing effects of EXCC on breast cancer cells were alleviated by NAC (Fig. 3B).

Fig. 3.

Fig. 3

Open in a new tab

Apoptotic effects of and caspase 3, 8, and 9 activation (Western blotting) by EXCC. (A) Annexin V apoptosis assay. Breast cancer (MCF7 and MDA-MB-231) and nonmalignant (M10) cells were incubated with EXCC for 48 h. Annexin V (+)/7ADD (+/Inline graphic) (%) is assigned as apoptosis (%). (B) Annexin V apoptosis assay (NAC/EXCC). After NAC 1 h pretreatment, cells were exposed to EXCC (50 µg/ml) for 24 and 48 h, i.e., NAC/EXCC. Non-overlapping letters reveal significant outcomes (p < 0.05). (C) Western blotting of cleaved caspases 3, 8, and 9 (c-Cas 3, 8, and 9). NAC/EXCC and ZVAD/EXCC indicate NAC 1 h and ZVAD 2 h pretreatment followed by EXCC (50 µg/ml) for 24 and 48 h. Non-overlapping letters reveal significant outcomes (p < 0.05). Data are expressed as means ± SD (n = 3). Notably, the blots were cut before hybridization with antibodies during blotting. After the first antibody was used to detect a target, some cut blots were stripped to detect the next target sequentially. The blots for c-cas 9, c-cas 8, and actin were the same. They were stripped sequentially for different antibodies (c-cas 9 -> c-cas 8 -> actin)

EXCC shows caspase 3-, 8-, and 9-activating effects (Western blotting)

The EXCC-triggered apoptosis assessed using Annexin V was further validated with Western blotting. Terminal apoptotic signaling, such as caspase 3 activation [35], was triggered by EXCC in breast cancer cells at 24 and 48 h (Fig. 3C). Extrinsic and intrinsic apoptotic signaling molecules, such as caspases 8 and 9 [35], were highly expressed. Furthermore, these caspase signaling-activated effects of EXCC were higher in breast cancer than in M10 cells and were attenuated by NAC and ZVAD.

EXCC shows caspase 3-, 8-, and 9-activating effects (flow cytometry)

The EXCC-activated apoptotic signaling (caspases 3, 8, and 9) was assessed using flow cytometry. Their activated (cleaved) forms were measured by generating fluorescence through the use of a commercial kit. Caspases 3, 8, and 9 were activated in breast cancer cells by EXCC at various concentrations (Fig. 4A, C, and E) and for different times (Fig. 4B, D and F). Their activation was higher in breast cancer than in M10 cells. Furthermore, these caspase signaling-activating effects of EXCC on breast cancer cells were attenuated by NAC.

Fig. 4.

Fig. 4

Open in a new tab

Activation of caspases 3, 8, and 9 by EXCC (flow cytometry). (A) Caspases 3, (C) caspase 8, and (E) caspase 9 activation assays. Breast cancer (MCF7 and MDA-MB-231) and nonmalignant (M10) cells were incubated with EXCC for 48 h. (+) inside the flow cytometry panel is assigned as the caspases 3, 8, and 9 (+) events. (B) Caspases 3, (D) caspase 8, and (F) caspase 9 activation assays of NAC/EXCC. After NAC 1 h pretreatment, cells were exposed to EXCC (50 µg/ml) for 24 and 48 h, i.e., NAC/EXCC. Non-overlapping letters reveal significant outcomes (p < 0.05). Data are expressed as means ± SD (n = 3)

EXCC shows ROS- and GSH-modulating effects

The oxidative stress inhibitor NAC attenuated several EXCC-induced responses. NAC, a cysteine-providing antioxidant, can replete GSH levels [36]. This warrants the validation of oxidative stress generation and GSH depletion upon EXCC treatment in breast cancer cells.

Using flow cytometry, ROS were found to be induced by EXCC in breast cancer cells at various concentrations and treatment times (Fig. 5A and B). The ROS levels were higher in breast cancer than in M10 cells. Furthermore, EXCC’s ROS-inducing effects in breast cancer cells were alleviated by NAC (Fig. 5B).

Fig. 5.

Fig. 5

Open in a new tab

ROS- and GSH-modulating effects of EXCC. (A) ROS and (C) GSH assay. Breast cancer (MCF7 and MDA-MB-231) and nonmalignant (M10) cells were incubated with EXCC for 48 h. (+) and (Inline graphic) are assigned as ROS (+) and GSH (Inline graphic) populations. (B) ROS and (D) GSH assay of NAC/EXCC. After NAC 1 h pretreatment, cells were exposed to EXCC (50 µg/ml) for 24 and 48 h, i.e., NAC/EXCC. Non-overlapping letters reveal significant outcomes (p < 0.05). Data are expressed as means ± SD (n = 3)

Moreover, GSH (Inline graphic) (%) represents the GSH depletion. Using flow cytometry, GSH (Inline graphic) (%) was induced by EXCC in breast cancer cells treated with various concentrations and for different times (Fig. 5C and D). The GSH (Inline graphic) (%) effect was higher in breast cancer than in M10 cells. Furthermore, EXCC’s GSH-depleting effects on breast cancer cells were attenuated by NAC (Fig. 5D).

EXCC shows MitoSOX- and MMP-modulating effects

In addition to ROS and GSH, MitoSOX and MMP [37, 38] can be used to measure the degree of oxidative stress in cells. MitoSOX were induced by EXCC in breast cancer cells at various concentrations and times (Fig. 6A and B). The MitoSOX levels were higher in breast cancer than in M10 cells. Furthermore, EXCC’s MitoSOX-inducing effects on breast cancer cells were alleviated by NAC (Fig. 6B).

Fig. 6.

Fig. 6

Open in a new tab

MitoSOX- and MMP-modulating effects of EXCC. (A) MitoSOX and (C) MMP assay. Breast cancer (MCF7 and MDA-MB-231) and nonmalignant (M10) cells were incubated with EXCC for 48 h. (+) and (Inline graphic) are assigned as MitoSOX (+) and MMP (Inline graphic) populations. (B) MitoSOX and (D) MMP assay of NAC/EXCC. After NAC 1 h pretreatment, cells were exposed to EXCC (50 µg/ml) for 24 and 48 h, i.e., NAC/EXCC. Non-overlapping letters reveal significant outcomes (p < 0.05). Data are expressed as means ± SD (n = 3)

Moreover, MMP (Inline graphic) (%) represents the degree of MMP depletion. Using flow cytometry, MMP (Inline graphic) (%) was induced by EXCC in breast cancer cells at various concentrations and times (Fig. 6C and D). The MMP (Inline graphic) (%) was higher in breast cancer than in M10 cells. Furthermore, EXCC’s MMP-depleting effects on breast cancer cells were attenuated by NAC (Fig. 6D).

EXCC shows DNA damage-inducing effects

γH2AX and 8-OHdG (+) (%) were used to measure DNA damage induced by EXCC in breast cancer cells treated with various concentrations (Fig. 7A and C) and for different times (Fig. 7B and D). The γH2AX and 8-OHdG levels were higher in breast cancer than in M10 cells. Furthermore, EXCC’s DNA-damaging effects on breast cancer cells were attenuated by NAC (Fig. 7B and D).

Fig. 7.

Fig. 7

Open in a new tab

DNA damage (γH2AX and 8-OHdG)-inducing effects of EXCC. (A) γH2AX and (C) 8-OHdG assay. Breast cancer (MCF7 and MDA-MB-231) and nonmalignant (M10) cells were incubated with EXCC for 48 h. (+) is assigned as γH2AX and 8-OHdG (+) populations. (B) γH2AX and (D) 8-OHdG assay of NAC/EXCC. After NAC 1 h pretreatment, cells were exposed to EXCC (50 µg/ml) for 24 and 48 h, i.e., NAC/EXCC. Non-overlapping letters reveal significant outcomes (p < 0.05). Data are expressed as means ± SD (n = 3)

Discussion

Our previous study demonstrated the EXCC’s antiproliferative effects against bladder cancer cells [21]. However, this anti-bladder cancer study did not consider the impact on normal cells. Therefore, the drug safety of EXCC remains unclear. The current study evaluated the anti-breast cancer effects of EXCC by examining the proliferation of TNBC, non-TNBC, and normal cells and exploring its anticancer molecular mechanisms.

In the 48 h ATP assay, the IC50 values of EXCC for breast cancer cells (MDA-MB-468, MDA-MB-231, MCF7, and Hs578t) were 26.07, 30.36, 33.8, and 36.43 µg/ml (Fig. 1A). This indicates that EXCC is effective in antiproliferation against TNBC and non-TNBC (MCF7) cells. Therefore, the antiproliferation mechanism of EXCC may be unrelated to common targets, such as ER, PR, and HER2. It warrants an advanced investigation of the potential targets of EXCC in the future.

In comparison, bladder cancer cells (BFTC905, T24, and 5637) exhibited IC50 values of EXCC ranging from 51 to 100 µg/ml, regarding the 48 h ATP assay [21]. These findings suggest that EXCC is more effective in treating breast than bladder cancer cells, regarding the same treatment time and proliferation assay. It also extends the anticancer application to breast cancer in addition to bladder cancer cells.

Furthermore, the EXCC effectiveness of the anti-bladder cancer study [21] did not compare with the clinical drugs. In the current study, cisplatin showed IC50 values of 13.76 and 20.69 µg/ml for MCF7 and MDA-MB-231 cells (Fig. 1A). Although cisplatin is slightly more effective than EXCC, cisplatin exhibits potential adverse effects in treating cancer patients [39]. Meanwhile, EXCC shows low cytotoxicity to normal breast cells (M10) (Fig. 1A). The selectivity indices of EXCC-treated MDA-MB-468, MDA-MB-231, MCF7, and Hs578t breast cancer cells are 7.62, 6.54, 5.88, and 5.45, respectively. Therefore, EXCC has the potential for application as a breast cancer therapy with minimal side effects.

Differential levels of oxidative stresses cause cell death or survival. Oxidative stress at low to moderate levels promotes cancer cell proliferation, while deleterious levels enhance cell death [40]. Cells survive when they tolerate oxidative stress, while cells die with oxidative stress overload [41]. Oxidative stress is generally higher in cancer than in normal cells. When drugs induce deleterious oxidative stress [42, 43], they may exceed the threshold of oxidative stress of cancer cells but are tolerated by normal cells [41]. Consequently, these oxidative-stress-modulating drugs may cause the preferential killing of cancer cells rather than normal cells.

Many marine natural product-derived anti-breast cancer chemicals exhibit differential effects between breast cancer and normal cells in terms of oxidative stress. A quinazoline derivative [44] and ilamycin E [45] induce higher ROS and antiproliferative effects in breast cancer than in normal cells. Similarly, EXCC induces higher antiproliferative effects and oxidative stress in breast cancer than in normal cells as measured by ATP content, ROS, MitoSOX, and MMP changes. These changes are attenuated by NAC, indicating that EXCC triggers preferential oxidative-stress-associated antiproliferative effects on breast cancer rather than normal cells.

Since NAC attenuates these oxidative stress responses, the source of EXCC-generating oxidative stress may derive from GSH depletion because NAC is the precursor of cellular GSH [46, 47]. This GSH downregulation in breast cancer cells was demonstrated in EXCC-treated breast cancer cells, causing oxidative stress. Although non-enzymic antioxidant such as GSH was assessed, the role of the enzymatic antioxidant pathway in downregulating ROS clearance effects [43] of the EXCC treatment could not be excluded and needed further examination.

Oxidative stress [48] and GSH depletion [49–52] trigger apoptosis. Since EXCC causes GSH depletion and oxidative stress, it also induces higher apoptotic responses in breast cancer than in normal cells as measured by subG1 accumulation, Annexin V increase, and caspases 3, 8, and 9 activation. These changes are attenuated by NAC, indicating that EXCC triggers oxidative-stress-dependent apoptosis in breast cancer cells.

Oxidative stress causes DNA damage [41]. γH2AX and 8-OHdG are overexpressed in EXCC-treated breast cancer cells, which is attenuated by NAC. This suggests that EXCC triggers oxidative stress-dependent DNA damage. Moreover, some chemicals, such as aldehydes, simultaneously induce DNA damage and inhibit DNA repair [53]. γH2AX is a marker for a DNA double-strand break, which is generally repaired by nonhomologous end joining (NHEJ) and homologous recombination (HR) [54]. Human oxoguanine glycosylase 1 (hOGG1), a DNA glycosylase, is responsible for the excision repair of 8-OHdG [55]. Nitric oxide enhances the inhibition of DNA repair, upregulating 8-OHdG, in cholangiocytes [56]. Therefore, an evaluation of the expression of HR, NHEJ, and hOGG1 in an EXCC breast cancer study is warranted in future investigations.

Consequently, the current study demonstrated that EXCC exhibited excessive oxidative stress (ROS/MitoSOX overproduction and MMP/GSH depletion), apoptosis induction (annexin V and caspase 3/8/9 activation), DNA damage (γH2AX and 8-OHdG), and antiproliferation in cancer cells compared to normal cells in the ROS-dependent molecular mechanisms.

Furthermore, since two breast cancer cell lines from different subtypes, such as TNBC (MDA-MB-231) and non-TNBC (MCF7), were used, the patterns of their effects warrant comparison. Notably, the TNBC subtype is known to have a high basal intracellular ROS level [57]. Similarly, the current study showed that ROS’s basal level (untreated control) was higher in MDA-MB-231 than in MCF7 cells. At highest dose of EXCC (50 µg/ml), the levels (%) of ROS (+) is 83.3 vs. 96.7, GSH (-) is 17.5 vs. 18.7 MitoSOX (+) is 94.1 vs. 89.3, MMP (-) is 36.2 vs. 20.0, γH2AX (+) is 36.9 vs. 26.7, and 8-OHdG (+) is 86.0 vs. 91.4 for MDA-MB-231 and MCF7 cells, respectively. These results suggest that some EXCC-modulated effects (MitoSOX (+), MMP (-), and γH2AX (+)) are higher in TNBC than non-TNBC cells, but some (induced ROS (+) and 8-OHdG (+)) are not.

Moreover, the highest dose of EXCC exhibits similar caspases 3, 8, and 9 (+) levels (%), i.e., 96.9 vs. 95.5, 93.4 vs. 92.6, and 92.5 vs. 95.7 for MDA-MB-231 and MCF7 cells. Although the IC50 values were similar (30.36 and 33.8 µg/ml), the apoptosis (annexin V) (+) (%) levels were lower in MDA-MB-231 than in MCF7 cells, i.e., 29.15 vs. 79.7. This result suggests that apoptosis cannot solely attribute to the antiproliferative effects of EXCC between TNBC and non-TNBC cells. The involvement of non-apoptotic cell death, such as necroptosis, autophagy, and ferroptosis, caused by EXCC cannot be excluded. Overall, the basal and EXCC-induced levels of ROS for MDA-MB-231 and MCF7 cells cannot thoroughly explain these differential responses between TNBC and non-TNBC cells.

Conclusions

The anticancer effects of EXCC against breast cancer cells were demonstrated in this study. EXCC exhibited preferential killing of breast cancer rather than normal cells, indicating good drug safety for future applications such as EXCC-clinical drug combination treatment and in vivo study. EXCC also induced higher oxidative stress and associated responses regarding ROS, such as apoptosis and DNA damage. GSH depletion further supported the hypothesis that EXCC induced oxidative stress. Consequently, EXCC was confirmed to demonstrate anti-breast cancer proliferation effects mediated through oxidative-stress-dependent mechanisms.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Material 1 (1.3MB, pdf)

Author contributions

Conceptualization, J.-P.S., J.-H.S., and H.-W.C.; data curation, C.-W.Y.; formal analysis, C.-W.Y.; methodology, W.L., S.-Y.Y., C.-H.Y., and F.-R.C.; supervision, J.-H.S. and H.-W.C.; writing—original draft, C.-W.Y., and H.-W.C.; writing—review and editing, J.-P.S. and H.-W.C. All authors have read and agreed to the published version of the manuscript.

Funding

This study was partly supported by funds from the Ministry of Science and Technology (MOST 111-2320-B-037-015-MY3), the Kaohsiung Medical University (KMU-DK(A)113003 and KMU-TB114009), the National Sun Yat-sen University-KMU Joint Research Project (#NSYSUKMU 112-P06), the Kaohsiung Medical University Hospital (KMUH108-8M37, KMUH109-9M35, and KMUH111-1M32), and the Kaohsiung Medical University Research Center (KMU-TC113A04).

Data availability

All data generated or analyzed during this study are included in this published article.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Jun-Ping Shiau and Che-Wei Yang contributed equally to this work.

Contributor Information

Jyh-Horng Sheu, Email: sheu@mail.nsysu.edu.tw.

Hsueh-Wei Chang, Email: changhw@kmu.edu.tw.

References

  • 1.Siegel RL, Giaquinto AN, Jemal A. Cancer statistics, 2024. CA Cancer J Clin. 2024;74(1):12–49. [DOI] [PubMed] [Google Scholar]
  • 2.Waks AG, Winer EP. Breast cancer treatment: A review. JAMA. 2019;321(3):288–300. [DOI] [PubMed] [Google Scholar]
  • 3.Tao JJ, Visvanathan K, Wolff AC. Long term side effects of adjuvant chemotherapy in patients with early breast cancer. Breast. 2015;24(Suppl 2):S149–153. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Tariq K, Rana F. TNBC vs. Non-TNBC: A five-year retrospective review of differences in mean age, family history, smoking history and stage at diagnosis at an inner City university program. World J Oncol. 2013;4(6):241–7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Goncalves H Jr., Guerra MR, Duarte Cintra JR, Fayer VA, Brum IV, Bustamante Teixeira MT. Survival study of triple-negative and non-triple-negative breast cancer in a Brazilian cohort. Clin Med Insights Oncol. 2018;12:1179554918790563. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Nathan MR, Schmid P. A review of fulvestrant in breast cancer. Oncol Ther. 2017;5(1):17–29. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Baselga J, Campone M, Piccart M, Burris HA 3rd, Rugo HS, Sahmoud T, Noguchi S, Gnant M, Pritchard KI, Lebrun F, et al. Everolimus in postmenopausal hormone-receptor-positive advanced breast cancer. N Engl J Med. 2012;366(6):520–9. [DOI] [PMC free article] [PubMed]
  • 8.Swain SM, Shastry M, Hamilton E. Targeting HER2-positive breast cancer: advances and future directions. Nat Rev Drug Discov. 2023;22(2):101–26. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Hou XM, Hai Y, Gu YC, Wang CY, Shao CL. Chemical and bioactive marine natural products of coral-derived microorganisms (2015–2017). Curr Med Chem. 2019;26(38):6930–41. [DOI] [PubMed] [Google Scholar]
  • 10.Sang VT, Dat TTH, Vinh LB, Cuong LCV, Oanh PTT, Ha H, Kim YH, Anh HLT, Yang SY. Coral and coral-associated microorganisms: A prolific source of potential bioactive natural products. Mar Drugs. 2019;17(8):468. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Alves C, Diederich M. Marine natural products as anticancer agents. Mar Drugs. 2021;19(8):447. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Khalifa SAM, Elias N, Farag MA, Chen L, Saeed A, Hegazy MF, Moustafa MS, Abd El-Wahed A, Al-Mousawi SM, Musharraf SG, et al. Marine natural products: A source of novel anticancer drugs. Mar Drugs. 2019;17(9):491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Islam MT, Hossain R, Hassan SMH, Salehi B, Martins N, Sharifi-Rad J, Amarowicz R. Biological activities of sinularin: A literature-based review. Cell Mol Biol (Noisy-le-grand). 2020;66(4):33–6. [PubMed] [Google Scholar]
  • 14.Wali AF, Majid S, Rasool S, Shehada SB, Abdulkareem SK, Firdous A, Beigh S, Shakeel S, Mushtaq S, Akbar I, et al. Natural products against cancer: review on phytochemicals from marine sources in preventing cancer. Saudi Pharm J. 2019;27(6):767–77. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Sheu JH, Sung PJ, Cheng MC, Liu HY, Fang LS, Duh CY, Chiang MY. Novel cytotoxic diterpenes, excavatolides A-E, isolated from the Formosan Gorgonian Briareum excavatum. J Nat Prod. 1998;61(5):602–8. [DOI] [PubMed] [Google Scholar]
  • 16.Sung PJ, Su JH, Wang GH, Lin SF, Duh CY, Sheu JH. Excavatolides F-M, new briarane diterpenes from the Gorgonian Briareum excavatum. J Nat Prod. 1999;62(3):457–63. [DOI] [PubMed] [Google Scholar]
  • 17.Lin YY, Lin SC, Feng CW, Chen PC, Su YD, Li CM, Yang SN, Jean YH, Sung PJ, Duh CY, et al. Anti-inflammatory and analgesic effects of the marine-derived compound excavatolide B isolated from the culture-type Formosan Gorgonian Briareum excavatum. Mar Drugs. 2015;13(5):2559–79. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Velmurugan BK, Yang HH, Sung PJ, Weng CF. Excavatolide B inhibits nonsmall cell lung cancer proliferation by altering peroxisome proliferator activated receptor gamma expression and PTEN/AKT/NF-Kbeta expression. Environ Toxicol. 2017;32(1):290–301. [DOI] [PubMed] [Google Scholar]
  • 19.Lin YY, Jean YH, Lee HP, Lin SC, Pan CY, Chen WF, Wu SF, Su JH, Tsui KH, Sheu JH, et al. Excavatolide B attenuates rheumatoid arthritis through the Inhibition of osteoclastogenesis. Mar Drugs. 2017;15(1):9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Chen HW, Liu FC, Kuo HM, Tang SH, Niu GH, Zhang MM, Tsou LK, Sung PJ, Wen ZH. Immunomodulatory and anti-angiogenesis effects of excavatolide B and its derivatives in alleviating atopic dermatitis. Biomed Pharmacother. 2024;172:116279. [DOI] [PubMed] [Google Scholar]
  • 21.Yang CW, Chien TM, Yen CH, Wu WJ, Sheu JH, Chang HW. Antibladder cancer effects of excavatolide C by inducing oxidative stress, apoptosis, and DNA damage in vitro. Pharmaceuticals (Basel). 2022;15(8):917. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Chien TM, Yang CW, Yen CH, Yeh BW, Wu WJ, Sheu JH, Chang HW. Excavatolide C/cisplatin combination induces antiproliferation and drives apoptosis and DNA damage in bladder cancer cells. Arch Toxicol. 2024;98(5):1543–60. [DOI] [PubMed] [Google Scholar]
  • 23.Hung JH, Chen CY, Omar HA, Huang KY, Tsao CC, Chiu CC, Chen YL, Chen PH, Teng YN. Reactive oxygen species mediate Terbufos-induced apoptosis in mouse testicular cell lines via the modulation of cell cycle and pro-apoptotic proteins. Environ Toxicol. 2016;31(12):1888–98. [DOI] [PubMed] [Google Scholar]
  • 24.Huang CH, Yeh JM, Chan WH. Hazardous impacts of silver nanoparticles on mouse oocyte maturation and fertilization and fetal development through induction of apoptotic processes. Environ Toxicol. 2018;33(10):1039–49. [DOI] [PubMed] [Google Scholar]
  • 25.Wang TS, Lin CP, Chen YP, Chao MR, Li CC, Liu KL. CYP450-mediated mitochondrial ROS production involved in Arecoline N-oxide-induced oxidative damage in liver cell lines. Environ Toxicol. 2018;33(10):1029–38. [DOI] [PubMed] [Google Scholar]
  • 26.Vignon C, Debeissat C, Georget MT, Bouscary D, Gyan E, Rosset P, Herault O. Flow cytometric quantification of all phases of the cell cycle and apoptosis in a two-color fluorescence plot. PLoS ONE. 2013;8(7):e68425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Shiau JP, Chuang YT, Yang KH, Chang FR, Sheu JH, Hou MF, Jeng JH, Tang JY, Chang HW. Brown algae-derived fucoidan exerts oxidative stress-dependent antiproliferation on oral cancer cells. Antioxid (Basel). 2022;11(5):841. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Fan HC, Hsieh YC, Li LH, Chang CC, Janouskova K, Ramani MV, Subbaraju GV, Cheng KT, Chang CC. Dehydroxyhispolon Methyl ether, a hispolon derivative, inhibits WNT/beta-catenin signaling to elicit human colorectal carcinoma cell apoptosis. Int J Mol Sci. 2020;21(22):8839. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Lee CH, Shih YL, Lee MH, Au MK, Chen YL, Lu HF, Chung JG. Bufalin induces apoptosis of human osteosarcoma U-2 OS cells through Endoplasmic reticulum stress, caspase- and mitochondria-dependent signaling pathways. Molecules. 2017;22(3):437. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Chou WH, Liu KL, Shih YL, Chuang YY, Chou J, Lu HF, Jair HW, Lee MZ, Au MK, Chung JG. Ouabain induces apoptotic cell death through caspase- and mitochondria-dependent pathways in human osteosarcoma U-2 OS cells. Anticancer Res. 2018;38(1):169–78. [DOI] [PubMed] [Google Scholar]
  • 31.Gambles MT, Li J, Wang J, Sborov D, Yang J, Kopecek J. Crosslinking of CD38 receptors triggers apoptosis of malignant B cells. Molecules. 2021;26(15):4658. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Liu T, Sun L, Zhang Y, Wang Y, Zheng J. Imbalanced GSH/ROS and sequential cell death. J Biochem Mol Toxicol. 2022;36(1):e22942. [DOI] [PubMed] [Google Scholar]
  • 33.Lu MC, Li TY, Hsieh YC, Hsieh PC, Chu YL. Chemical evaluation and cytotoxic mechanism investigation of Clinacanthus nutans extract in lymphoma SUP-T1 cells. Environ Toxicol. 2018;33(12):1229–36. [DOI] [PubMed] [Google Scholar]
  • 34.Sgarbi G, Gorini G, Liuzzi F, Solaini G, Baracca A. Hypoxia and IF(1) expression promote ROS decrease in cancer cells. Cells. 2018;7(7):64. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Boice A, Bouchier-Hayes L. Targeting apoptotic caspases in cancer. Biochim Biophys Acta Mol Cell Res. 2020;1867(6):118688. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Giustarini D, Milzani A, Dalle-Donne I, Rossi R. How to increase cellular glutathione. Antioxidants. 2023;12(5):1094. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Maharjan S, Oku M, Tsuda M, Hoseki J, Sakai Y. Mitochondrial impairment triggers cytosolic oxidative stress and cell death following proteasome Inhibition. Sci Rep. 2014;4:5896. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Kim SH, Kim H. Inhibitory effect of Astaxanthin on oxidative stress-induced mitochondrial dysfunction- A mini-review. Nutrients. 2018;10(9):1137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Aldossary SA. Review on Pharmacology of cisplatin: clinical use, toxicity and mechanism of resistance of cisplatin. Biomed Pharmacol J. 2019;12(1):7–15. [Google Scholar]
  • 40.Nakamura H, Takada K. Reactive oxygen species in cancer: current findings and future directions. Cancer Sci. 2021;112(10):3945–52. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Tang JY, Ou-Yang F, Hou MF, Huang HW, Wang HR, Li KT, Fayyaz S, Shu CW, Chang HW. Oxidative stress-modulating drugs have Preferential anticancer effects - involving the regulation of apoptosis, DNA damage, Endoplasmic reticulum stress, autophagy, metabolism, and migration. Semin Cancer Biol. 2019;58:109–17. [DOI] [PubMed] [Google Scholar]
  • 42.Teppo HR, Soini Y, Karihtala P. Reactive oxygen species-mediated mechanisms of action of targeted cancer therapy. Oxid Med Cell Longev. 2017;2017:1485283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Wang J, Sun D, Huang L, Wang S, Jin Y. Targeting reactive oxygen species capacity of tumor cells with repurposed drug as an anticancer therapy. Oxid Med Cell Longev. 202;2021:8532940. [DOI] [PMC free article] [PubMed]
  • 44.De AK, Muthiyan R, Mondal S, Mahanta N, Bhattacharya D, Ponraj P, Muniswamy K, Kundu A, Kundu MS, Sunder J, et al. A natural Quinazoline derivative from marine sponge Hyrtios erectus induces apoptosis of breast cancer cells via ROS production and intrinsic or extrinsic apoptosis pathways. Mar Drugs. 2019;17(12):658. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Zhou W, Fang H, Wu Q, Wang X, Liu R, Li F, Xiao J, Yuan L, Zhou Z, Ma J, et al. Ilamycin E, a natural product of marine actinomycete, inhibits triple-negative breast cancer partially through ER stress-CHOP-Bcl-2. Int J Biol Sci. 2019;15(8):1723–32. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Jasek-Gajda E, Jurkowska H, Jasinska M, Lis GJ. Targeting the MAPK/ERK and PI3K/AKT signaling pathways affects NRF2, Trx and GSH antioxidant systems in leukemia cells. Antioxid (Basel). 2020;9(7):633. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Rostila AM, Anttila SL, Lalowski MM, Vuopala KS, Toljamo TI, Lindstrom I, Baumann MH, Puustinen AM. Reactive oxygen species-regulating proteins Peroxiredoxin 2 and thioredoxin, and glyceraldehyde-3-phosphate dehydrogenase are differentially abundant in induced sputum from smokers with lung cancer or asbestos exposure. Eur J Cancer Prev. 2020;29(3):238–47. [DOI] [PubMed] [Google Scholar]
  • 48.Redza-Dutordoir M, Averill-Bates DA. Activation of apoptosis signalling pathways by reactive oxygen species. Biochim Biophys Acta. 2016;1863(12):2977–92. [DOI] [PubMed] [Google Scholar]
  • 49.Franco R, DeHaven WI, Sifre MI, Bortner CD, Cidlowski JA. Glutathione depletion and disruption of intracellular ionic homeostasis regulate lymphoid cell apoptosis. J Biol Chem. 2008;283(52):36071–87. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Schnelldorfer T, Gansauge S, Gansauge F, Schlosser S, Beger HG, Nussler AK. Glutathione depletion causes cell growth Inhibition and enhanced apoptosis in pancreatic cancer cells. Cancer. 2000;89(7):1440–7. [PubMed] [Google Scholar]
  • 51.Petricca S, Celenza G, Luzi C, Cinque B, Lizzi AR, Franceschini N, Festuccia C, Iorio R. Synergistic activity of ketoconazole and miconazole with Prochloraz in inducing oxidative stress, GSH depletion, mitochondrial dysfunction, and apoptosis in mouse Sertoli TM4 cells. Int J Mol Sci. 2022;23(10):5429. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52.Sang J, Li W, Diao HJ, Fan RZ, Huang JL, Gan L, Zou MF, Tang GH, Yin S. Jolkinolide B targets thioredoxin and glutathione systems to induce ROS-mediated paraptosis and apoptosis in bladder cancer cells. Cancer Lett. 2021;509:13–25. [DOI] [PubMed] [Google Scholar]
  • 53.Weng MW, Lee HW, Park SH, Hu Y, Wang HT, Chen LC, Rom WN, Huang WC, Lepor H, Wu XR, et al. Aldehydes are the predominant forces inducing DNA damage and inhibiting DNA repair in tobacco smoke carcinogenesis. Proc Natl Acad Sci U S A. 2018;115(27):E6152–61. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54.Seluanov A, Mao Z, Gorbunova V. Analysis of DNA double-strand break (DSB) repair in mammalian cells. J Vis Exp. 2010(43):e2002. [DOI] [PMC free article] [PubMed]
  • 55.Shinmura K, Yokota J. The OGG1 gene encodes a repair enzyme for oxidatively damaged DNA and is involved in human carcinogenesis. Antioxid Redox Signal. 2001;3(4):597–609. [DOI] [PubMed] [Google Scholar]
  • 56.Jaiswal M, LaRusso NF, Shapiro RA, Billiar TR, Gores GJ. Nitric oxide-mediated Inhibition of DNA repair potentiates oxidative DNA damage in cholangiocytes. Gastroenterology. 2001;120(1):190–9. [DOI] [PubMed] [Google Scholar]
  • 57.Sarmiento-Salinas FL, Delgado-Magallon A, Montes-Alvarado JB, Ramirez-Ramirez D, Flores-Alonso JC, Cortes-Hernandez P, Reyes-Leyva J, Herrera-Camacho I, Anaya-Ruiz M, Pelayo R, et al. Breast cancer subtypes present a differential production of reactive oxygen species (ROS) and susceptibility to antioxidant treatment. Front Oncol. 2019;9:480. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1 (1.3MB, pdf)

Data Availability Statement

All data generated or analyzed during this study are included in this published article.

Articles from BMC Cancer are provided here courtesy of BMC

RESOURCES