Curcumin-like structure (CCA-1.1) induces permanent mitotic arrest (Senescence) on Triple-negative breast cancer (TNBC) cells, 4T1

 

Dhania Novitasari¹, Riris Istighfari Jenie^1,2, Febri Wulandari¹, Rohmad Yudi Utomo^1,3,

Dyaningtyas Dewi Pamungkas Putri^1,4, Jun-ya Kato⁵, Edy Meiyanto^1,2*

¹Cancer Chemoprevention Research Center, Faculty of Pharmacy, Universitas Gadjah Mada (UGM),

Sekip Utara, Yogyakarta 55281, Indonesia.

²Macromolecular Engineering Laboratory, Department of Pharmaceutical Chemistry,

Faculty of Pharmacy UGM, Sekip Utara, Yogyakarta 55281, Indonesia.

³Medicinal Chemistry Laboratory, Department of Pharmaceutical Chemistry,

Faculty of Pharmacy, UGM, Sekip Utara, Yogyakarta 55281, Indonesia.

⁴Pharmacology and Toxicology Laboratory, Department of Pharmacology and Clinical Pharmacy,

Faculty of Pharmacy, Universitas Gadjah Mada, 55281, Indonesia.

⁵Laboratory of Tumor Cell Biology, Nara Institute of Science and Technology, Ikoma, Nara, Japan.

 

ABSTRACT:

Triple-negative breast cancer (TNBC) remains as the deadliest cancer type due to the lack of treatment options. Hence, several attempts have been made to develop new anticancer for TNBC therapy. This study intended to challenge curcumin analog (CCA)-1.1, which is derived from pentagamavunone-1 structure, against the 4T1 cell line and TNBC cell model, covering the cytotoxic activity in correlation with cell cycle progression, apoptosis induction, reactive oxygen species (ROS) generation, and senescence evidence. The cell viability, cell cycle profile, apoptosis induction, intracellular ROS level, and senescence induction were determined in vitro using trypan blue exclusion, propidium iodide (PI) staining, Annexin-PI staining, dichlorofluorescein diacetate staining, and senescence-associated-β-gal method. CCA-1.1 showed cytotoxic activity on 4T1 cells, giving half maximal inhibitory concentration value of 3mM, but was less toxic on non-cancerous 3T3-L1 cells. CCA-1.1 induced rapid cell death and inhibited cell cycle progression at the mitotic phase. Instead, of causing apoptosis, CCA-1.1 induced mitotic catastrophe. Furthermore, CCA-1.1 itself increased the intracellular ROS level and induced senescence, possibly through catastrophic cell death. Altogether, our preliminary study strengthens the potency of CCA-1.1 for its anticancer activities against TNBC cells and prospective to be pharmaceutically developed as a novel candidate for cancer therapy.

 

 

 

INTRODUCTION:

Among the subtypes of breast cancers, triple-negative breast cancer (TNBC) needs serious attention for the development of an effective and safe therapeutic system. TNBC subtype accounts for 10%–20% of diagnosed cases, with severe prognosis due to the difficulties of treatment and the high metastatic characteristic of this cancer subtype^1–5.

 

TNBC lacks hormones and growth factor receptor [estrogen receptor (ER)⁻, progesterone receptor-negative, and human epidermal growth factor receptor 2 (HER2)⁻ expression⁶, thus, this cancer subtype is unsuitable for endocrine- or HER2-targeted drugs⁷, such as tamoxifen for the ER⁺ subtype or trastuzumab for the HER2⁺ subtype^8–10. The standard therapeutic approach for most TNBC cases is the use of broad-spectrum cytotoxic chemotherapeutic agents^11–14, surgery, and radiotherapy^11,15–17. However, around 60% of TNBC patients are non-responsive during these treatments ^7,18,19. Hence, several attempts and developments should be made to find novel chemotherapeutic choices for TNBC.

Currently, several options are available for chemotherapy for TNBC, mainly targeting phosphoinositide 3-kinase pathway inhibitors, cyclin-dependent kinase inhibitors, and immune checkpoint inhibitors^18,20. The chemotherapeutic drugs commonly used to treat TNBC, such as anthracyclins, taxanes, and vinca alkaloids, cause inevitable side effects and worsen patients’ condition because they are not explicitly targeted in cancer cells^21,22. Recently, US Food and Drug Administration approved an humanized monoclonal antibody named sacituzumab govitecan-hziy, which targets human trophoblast cell-surface antigen 2, as a treatment for patients with metastasized TNBC^23,24. Nevertheless, these drugs are costly because they require an antibody for their delivery. Furthermore, the side effects that arise from the use of these drugs are still under investigation. As a consequence, the development of new compounds that uniquely target cell cycle progression and interfere with cancer cell metabolism, leading to cancer cell death, with minimal side effects and low chance of relapse would be beneficial to the effective treatment of TNBC.

 

The curcumin analog (CCA) that we developed in 2003, that is, pentagamavunone-1 (PGV-1) or 2,5-bis-(4-hydroxy-3,5-dimethyl-benzylidene)cyclo-pentanone, showed anticancer activity against TNBC subtype through in vitro and in vivo studies. PGV-1 effectively inhibited 4T1 (a murine-derived TNBC cell line) cell proliferation (half maximal inhibitory concentration (IC₅₀) <5µM) with a proposed mechanism including G2/M cell cycle arrest and induced cell senescence by increasing the intracellular reactive oxygen species (ROS) level²⁵. The proliferation inhibition PGV-1 was irreversible against K562 leukemia cells²⁶. Furthermore, PGV-1 inhibited the ROS-metabolic enzymes, which eventually elevated ROS level and caused cell senescence. An in vivo study showed the importance of developing this compound and revealed that oral administration of PGV-1 effectively inhibited tumor development but resulted in negligible side effects^26,27. Recently, a novel synthetic CCA, named 2,5-bis-(4-hydroxy-3,5-dimethyl benzylidene)-cyclopentanol (CCA-1.1), was synthesized with modification based on PGV-1 structure (Figs. 1A and 1B) to achieve enhanced stability and solubility compared with PGV-1 and curcumin. Through in vitro studies, CCA-1.1 exhibited better cytotoxic effect than PGV-1 against cancer cells but remained less toxic toward healthy cells. Furthermore, CCA-1.1 performed better than PGV-1 in a molecular docking study, showing a higher binding affinity toward several ROS scavengers through the formation of a hydrogen bond from CCA-1.1 and its hydroxyl group, which might have also partly contributed to the high intracellular ROS level²⁸ Therefore, CCA-1.1 is valuable for further investigation of its action against the 4T1 cell line with focus on several effects, including cell cycle modulation, apoptosis, cell senescence, and intracellular ROS level.

 

MATERIAL AND METHODS:

Chemical compounds:

CCA-1.1 and PGV-1 (2,5-bis-(4-hydroxy-3,5-dimethylbenzylidene)-cyclopentanone) were collected from Cancer Chemoprevention Research Center, Universitas Gadjah Mada. Doxorubicin hydrochloride (Dox) powder was purchased from Sigma.

 

Cells culture and propagation:

We used 4T1 cells (gifted by Prof. Masashi Kawaichi, MD. PhD. from NAIST, Japan) as a model for TNBC and 3T3-L1 cells (kindly provided by Dr. Rer. Nat. Muhammad Hasan Bashari and Dr. M. Kes from Universitas Padjajaran) throughout this study. The 4T1 and 3T3-L1 cells were maintained in high-glucose Dulbecco’s Modified Eagle Medium (Gibco Life Technologies, CA, USA) added with fetal bovine serum (10% v/v) (Gibco Life Technologies, CA, USA), HEPES (Sigma Aldrich, MO, USA), sodium bicarbonate (Sigma Aldrich, MO, USA), penicillin (150IU/mL), and streptomycin (150µg/mL) (Gibco Life Technologies, CA, USA) and then stored at 37°C incubator with 5% CO₂. After reaching confluence, the cells were detached with trypsin–ethylenediaminetetraacetic acid (Gibco Life Technologies, CA, USA) and sub-cultured in a tissue culture dish (Iwaki, Japan) or seeded in the well plate for further treatment.

 

Cytotoxic activity using trypan blue exclusion assay:

The cells were seeded in a 24-well plate (2×10⁴ cells/well) (Iwaki, Japan) and split into untreated and treated groups. The concentration variances of CCA-1.1 and PGV-1 as control were diluted within the culture medium. After 24 h, the medium was discarded, and the cells were washed with phosphate-buffered saline (PBS) 1× (Sigma Aldrich, MO, USA). The cells were then detached using trypsin and collected as cell suspension. Briefly, 10µL suspension was mixed with 10µL trypan blue (0.4%) (Sigma Aldrich, MO, USA) and incubated for 3 min at room temperature before counting the viable cells under an inverted microscope (Olympus CKX41). The quantified viable cells were plotted into concentration versus % viable cells to obtain the linear regression and converted into IC₅₀ value by using Excel MS Office 2016.

 

Cell cycle assay:

We assessed the cell cycle distribution of tested compounds by flow cytometry. Cells were grown in 6-well plate 24 h prior from the treatment with samples. The next day, all the medium were discarded and collected in each conical, the cells were trypsinized, and centrifuged 2,000rpm for 3 minutes. The collected cell pellets were then fixed for 30 minutes with ethanol. Then, the cells were washed twice with cold PBS and centrifuged before resuspended in propidium iodide solution (Invitrogen) (50μg/ml in PBS containing 1% triton X-100) with DNase-free RNase A (20μg/ml) for 15 minutes at 37°C in a dark place. Treated cells were then subjected into flow cytometer (BD Biosciences). The red fluorescence was measured using the default setting (log mode) after the cell debris was electronically gated out. Twenty thousand events were acquired for subsequent analyzed for each cell cycle phase with in-house program (BD Biosciences).

 

Apoptosis assay:

Apoptosis induction was determined through flow cytometry-based assay using Annexin-V-FLUOS staining kit. Briefly, 4T1 cells (2.5 × 10⁵) were planted into each well and incubated overnight before the treatment with either CCA-1.1, Dox, or its combination. After 24 h, the cells were detached and centrifuged before staining with Annexin-V-FLUOS staining kit (BD Biosciences, CA, USA) following the manufacturer’s instruction. We incubated the cell suspension for 10 min in a darkroom and ejected them into BD Accuri C6 flow cytometer (BD Bioscience, CA, USA). A total of 20,000 events were acquired and analyzed by in-house software, and the graph profile was visualized by Excel MS Office 2016.

 

Intracellular ROS level measurement:

We used flow cytometry-based assay using dichlorofluorescein diacetate (DCFDA) staining as described in the work of Meiyanto et al²⁵. The cells (5 × 10⁴) were planted in a 24-well plate and incubated overnight. After the detachment by using trypsin, the cell suspension was added with 375µL 1× supplemented buffer and stained by 20µM DCFDA (Sigma Aldrich, MO, USA) before another 30 min of incubation. The cell suspensions were added with 100nM Dox (positive control) or CCA-1.1 at various concentrations (treatment) and then incubated (37°C; CO₂ 5% incubator) for 4 h. The ROS level was determined with a flow cytometer with an excitation wavelength set at Ex 485nm/Em 535nm. The fluorescence intensity was quantified as fold change of the untreated group using Excel MS Office 2016.

 

Senescence assay:

The senescence in cancer cells was observed through senescence-associated (SA)-β-gal assay as mentioned in the work of Ahlina et al²⁹. The cells (1.2 × 10⁵) were planted in a six-well plate and treated with CCA-1.1 (0.5 and 1µM) for 24 h. A fixation solution (which contained 4% formaldehyde) was added to the cells for 20 min at room temperature. Then, the cells were washed again with PBS 1× and stained by using the X-gal solution. We observed the cells under an inverted microscope (200× magnification) during 3 days of incubation. The green-blue cells were marked as positive senescent cells and quantified using ImageJ software.

 

Statistical analysis:

The data collected in this study were presented as the mean of three data ± standard error (SE), followed by statistical analysis using Student’s t-test. All the p-values were included in each experiment figure.

 

RESULT:

Cytotoxic effect of CCA-1.1 against 4T1 cells:

This study intended to investigate the anticancer activities of CCA-1.1 against TNBC cells²⁸ reported that CCA-1.1 exhibited superior cytotoxic activity against several cancer cells, including 4T1 cells as a representative for TNBC cells. Therefore, we retested the cytotoxic effect of CCA-1.1 on these cells and observed a strong anti-proliferative activity comparable to that of PGV-1, with the IC₅₀ values of 3 and 4µM (Fig. 1C). We also tested the selectivity of CCA-1.1 to normal cells by using non-cancerous 3T3-L1 cells, which were derived from Swiss murine (Morrison and McGee, 2015). The 3T3-L1-treated cells showed a high percentage of viable cells after 24 h treatment with the compound up to a dose of 10 µM, achieving a selectivity index value greater than 2. Thus, CCA-1.1 has good selectivity toward cancer cells.

 

Figure 1. Chemical structure of (A) CCA-1.1 and (B) PGV-1, which were used to treat 4T1 breast cancer cells and 3T3-L1 (fibroblast-like) cells. Cells were planted in each well and treated with CCA-1.1 (A) or PGV-1. Cell viability was measured through trypan blue staining as mentioned in Methods. The cytotoxicities of CCA-1.1 and PGV-1 are presented as percentage of viable (mean ± SE) 4T1 cancer (C) and 3T3-L1 cells (D) and their IC₅₀ value (E).

Figure 2: CCA-1.1 induces cell cycle arrest in hyperploid formation of 4T1 cells. CCA-1.1 (1.5 and 3 µM) and Dox (100 nM; as control group) were treated for 24 h. Cell cycle phase was determined through PI staining and analyzed in a flow cytometer. (B) Cell distribution in each phase was presented as the mean from the percentage of total cells per phase ± SE (n = 3).

 

Effect of CCA-1.1 treatment on cell cycle modulation

The effect of PGV-1 on cell cycle progression is a characteristic of several cancer cell types, including 4T1 cells that induce G2/M and hyperploid cell accumulation ^25,30,31. We investigated the effect of CCA-1.1 on the cell cycle modulation in 4T1 cells. CCA-1.1 (1.5 and 3 µM) caused cell arrest at mitosis and in the subG1 population (Fig. 2). We suggest that hyperploid formation and sub-G1 accumulation (suspected as apoptosis) were the subsequent events in the response to cell cycle interruption and failure of cells to complete mitosis or not undergo complete arrest. In this cell cycle analysis, we used Dox as a marker because it can arrest cells at the G2/M phase³². Interestingly, the combination treatment (CCA-1.1 and Dox) using the respective concentrations of the cells exhibited cell cycle arrest at the G2/M phase, in a manner relatively similar to the Dox effect without hyperploid cell formation. These results indicate that CCA-1.1 and Dox provided distinct target mechanisms on cell cycle progression, in which CCA-1-1 acted at the pass-over of Dox. Thus, Dox acts at the upstream of G2/M phase (particularly in G2 phase), whereas CCA-1.1 acts at the downstream of the Dox target. Hence, the effect of Dox occurred prior to CCA-1.1, and the subsequent events caused by CCA-1.1 did not ensue. Perhaps, in this cell line, CCA-1.1 has the same target with PGV-1 on K562, which was demonstrated as the interruption of the cell cycle at prometaphase²⁶. This finding is an interesting phenomenon that should be explored further.

 

CCA-1.1 induces apoptosis of 4T1 cells

The cell cycle progression of 4T1 was interrupted at different phases by Dox and CCA-1.1, with the subG1 population suspected to be in apoptosis. We then clarified whether apoptosis occurred in the subsequent cell cycle arrest. Our findings showed that CCA-1.1 (1.5 and 3 µM) increased the percentage of apoptotic cells compared with untreated cells by 13% and 18%, respectively (Fig. 4). A single treatment with chemotherapy using Dox induced apoptosis and necrosis in 4T1 cells. However, the treatment with CCA-1.1 together with Dox induced more cells to undergo apoptosis rather than necrosis. The result is also in accordance with the cell cycle profile of CCA-1.1, which showed the accumulation of arrested cells (~20%) in subG1 phase (Fig. 3). Thus, CCA-1.1 induces cell death in correlation to apoptosis against 4T1 cells.

 

 

Figure 3: Treatment with CCA-1.1 induces apoptosis of 4T1 cancer cells. (A) Cells were treated with CCA-1.1 and Dox (100 nM), incubated for 20 h. The cells were stained with Annexin-PI before flow cytometry and analyzed for 20000 cells. (B) The percentage of cells was presented as mean ± SE (n = 3).

 

Figure 4. CCA-1.1 elevates the ROS level in 4T1 cells. (A) Cells were stained for DCFDA analysis and incubated before the treatment with either CCA-1.1 or Dox as a positive control for 4 h, subjected to flow cytometry, and analyzed for the intensity of fluorescence of oxidized DCFDA. (B) Fluorescence from measurement was converted into fold of untreated group and presented as mean ± SE (n = 3).

 

CCA-1.1 elevates ROS level in 4T1 cells

The intracellular ROS level is one of the crucial factors affecting apoptosis. The elevated level of intracellular ROS induces cell senescence and leads to apoptosis^33,34. We performed DCFDA assay to determine the intracellular ROS level in cells. Given the weak fluorescence of the treated compound, we normalized the measurement with an untreated group for every treatment. Our results showed that CCA-1.1 increased the ROS level in 4T1 cancer cell model after 4 h of treatment (Fig. 4). CCA-1.1 also induced ROS level more rapidly compared with Dox.

 

CCA-1.1 induces senescence in 4T1 cells

Given that treatment of CCA-1.1 induced high amount of intracellular ROS level in 4T1 cells and accumulation of cells in the subG1 phase, we evaluated whether CCA-1.1 can also induce senescence, a condition when cell cycle is arrested permanently and occurs in response to various forms of cellular stress³⁵ in metastatic cancer. We used SA-β-gal assay to determine the effect of CCA-1.1 (0.5 and 1 µM) on the senescence of 4T1 cells. CCA-1.1 induced senescence in up to 20% of total 4T1 cells (Fig. 5), suggesting that the cytotoxic effect of CCA-1.1 is also possibly mediated by senescence induction.

 

Figure 5: CCA-1.1 induces senescence in 4T1 cells. (A) Cells were treated with CCA-1.1 (0.5 and 1 µM) and Dox (100 nM) as positive control for 24 h before staining with X-gal solution. The fixated cells were then monitored by a light microscope and documented using a camera in three days of incubation. Arrows mark the senescent cells. (B) The percentage of senescent cells from each group was calculated and presented as mean ± SE from three different fields of view. The treatment group was tested for its significance against the untreated group by T-test with p-value as stated in the figure.

 

DISCUSSION:

The poor prognosis and the lack of proper chemotherapeutic drugs to treat breast cancer, particularly in TNBC, urged us to find an ideal anticancer candidate with multiple targets. In this study, we explored CCA-1.1, an analog of curcumin modified from PGV-1 structure, and demonstrated its strong anticancer effect against 4T1 cancer cells. However, CCA-1.1 showed less toxicity on fibroblast-like cells. CCA-1.1 notably stimulated cell cycle arrest in the polyploidy phase. Thus, the cells might fail to separate during mitosis and become hyperploids. Interestingly, the cell percentage in the sub-G1 population at the concentration of 1.5 µM CCA-1.1 was higher than that under 3 µM CCA-1.1, whereas the apoptotic cell number was higher during treatment with high-dose CCA-1.1. These data suggest that CCA-1.1 in high concentrations may induce rapid cell death. Therefore, this compound could not be observed in sub-G1 peak through cell cycle analysis. Although the CCA-1.1 and Dox combination induced apoptosis, the percentage in the sub-G1 phase in combination treatment was lower than in single treatment with CCA-1.1. The cells that accumulated in the sub-G1 phase consist of reduced DNA content, an essential characteristic of apoptotic cells³⁶. However, the apoptotic cells can also originate from the asymmetric division of polyploid cells, which activates signals to trigger apoptosis³⁷. CCA-1.1 possibly induces mitotic catastrophe, which is identified by giant micro-nucleated cells, reflects the aberrant separation of chromosomes ^38,39, and contains a near-tetraploid chromosome number⁴⁰.

 

Mitotic catastrophe allows, to a certain extent, the common features that occur with apoptosis: the cytochrome C release from mitochondria and externalization of phosphatidylserine (PS) in the plasma membrane⁴¹. Hence, Annexin in apoptosis assay also detected the translocated PS in the membrane surface during the catastrophic process in diploid cells during mitosis phase and caused failure in cytokinesis. This explanation was confirmed during the combination treatment with 100 nM Dox, which showed that the cell percentage in sub-G1 phase was lower compared with that in single treatment with CCA-1.1, because the cells were arrested at G2 phase in the presence of Dox, as reported by Lee et al.⁴². A low concentration of Dox triggered the depletion of several mitosis regulatory proteins and contributed to the aberrant nuclear division and subsequent cell death in the mitotic catastrophe. This phenomenon might also support the result obtained beforehand. The mechanism of cell death through mitotic catastrophe occurred after several days, whereas apoptosis transpired rapidly within hours⁴¹. Therefore, these data need to be assessed to clarify the phenomenon by using different strategies, for example, by modifying the time course during observation to find the precise target of CCA-1.1, which differs from other existing anticancer drugs.

 

CCA-1.1 escalated the intracellular ROS levels in cells, and this occurrence might have led to the increased number of senescent cells during the treatment with low-dose CCA-1.1. The ROS level can be elevated by the downregulation of p21 protein, which also causes senescence, leading to ROS generation and triggering catastrophe and mitotic arrest in cancer cells^43,44. The morphological-marker mitotic catastrophic process also promotes or occurs in parallel with cellular senescence, and in certain cases, the defects cause mitotic exit and induce cell senescence through mitotic catastrophe^45,46 regardless of the p53 status³⁹. New findings also revealed that cells can enter senescence as a tetraploid (4N DNA content)^47,48, and this event possibly occurred with CCA-1.1 treated cells, which were arrested in mitosis. We used a low dose of Dox (100 nM) to trigger stress-induced premature senescence in cells⁴⁹. In other studies, low concentration of Dox (15–120 ng/ml) induced cell senescence during the mitotic catastrophe and caused a major loss in the integrity of cellular membranes, whereas a high dose of Dox induced apoptosis⁴¹. Most cases of drug-induced senescence occurred with p53 activation pathway (p53-dependent)⁵⁰, and in the case of Dox, its low concentration induced senescence, whereas those with deficient p53 underwent apoptosis⁵¹. Given that 4T1 cells are phenotypically characterized as null-p53 cancer cells⁵², the low concentration of Dox led to the temporary formation of senescence-like phenotypes, mitotic catastrophe, and cell death. From this explanation, we suggest that Dox overtook CCA-1.1 in the catastrophic event in G2 phase and caused cell death, whereas CCA-1.1 itself was powerful enough to induce senescence through a catastrophic event, restrain cell cycle progression in mitosis phase, and rapidly induce cell death. However, the mechanism of this cell death remains unknown and should be determined in future studies.

 

Unlike the CCA-1.1, which still allows cells to replicate their DNA, Dox targets topoisomerase II⁵³ and disrupts DNA replication. However, a low concentration of Dox may induce the formation of multiple micronuclei cells and trigger cell death after mitotic catastrophe given the failure of cells to separate during cytokinesis. The results of the combination treatment of CCA-1.1 with Dox could not be observed after 72 h because most cells died during treatment. Still, we should conduct further investigations to explore and confirm the effect of CCA-1.1 activities on intracellular ROS levels for an extended incubation period and determine how cancer cells enter senescence from the mitosis phase after treatment with CCA-1.1.

Given these results, we highlighted that CCA-1.1 targets the cell cycle disruption, specifically during mitosis, promotes high ROS level, and induces senescence in 4T1 cells. This research is a preliminary study on the anticancer activity of CCA-1.1. Thus, all the possible mechanistic actions of CCA-1.1 should be addressed in the next study. Analysis by using time-course observation should be carried out to scrutinize the exact target of CCA-1.1 in cancer cells. Altogether, CCA-1.1 induces rapid cell death through cell cycle arrest in mitosis and the formation of hyperploid cells and causes mitotic catastrophe. The escalated intracellular ROS level induced by CCA-1.1 also contributed to the senescence phenomenon, which also led to cell death. However, other targets related to hyperploid cells and interaction with ROS scavengers should be explored further as potential targets for CCA-1.1. Overall, we propose CCA-1.1 as a novel candidate to be developed in the pharmaceutical industry as chemotherapeutic and co-chemotherapeutic agent against breast cancer, especially TNBC.

 

ACKNOWLEDGMENT:

We thank Master Education Leading to Doctoral Program for Excellent Graduate (PMDSU) Research Scheme, that granted this study (Contract Number: 6302/UN1/DITLIT/DIT-LIT/LT/2019) and Iida Foundation for supporting the experiments in NAIST, Japan. The authors are also thankful to Dr. Apt. Sari Haryanti, M. Si. from B2P2TOOT Tawangmangu, Indonesia, for providing laboratory facilities to carry out flow cytometry assays.

 

CONFLICT OF INTEREST:

All of the authors confirmed that we disclosed all of conflicts of interest in this study.

 

REFERENCES:

1.      De Santis CE, Fedewa SA, Goding Sauer A, Kramer JL, Smith RA, Jemal A. Breast cancer statistics, 2015: Convergence of incidence rates between black and white women: Breast Cancer Statistics, 2015. CA: A Cancer Journal for Clinicians. 2016; 66(1): 31-42. doi:10.3322/caac.21320

2.      Kohler BA, Sherman RL, Howlader N, et al. Annual Report to the Nation on the Status of Cancer, 1975-2011, Featuring Incidence of Breast Cancer Subtypes by Race/Ethnicity, Poverty, and State. J Natl Cancer Inst. 2015; 107(6). doi:10.1093/jnci/djv048

3.      Plasilova ML, Hayse B, Killelea BK, Horowitz NR, Chagpar AB, Lannin DR. Features of triple-negative breast cancer. Medicine (Baltimore). 2016;95(35). doi:10.1097/MD.0000000000004614

4.      Hariramani N, Jayanthi S. A Systematic Review of Intrinsic Factors and its Influence in Breast Cancer. Research Journal of Pharmacy and Technology. 2018;11(8): 3543-3546. doi:10.5958/0974-360X.2018.00654.6

5.      Asaad RA, Abdullah SS. Breast Cancer Subtypes (BCSs) Classification according to Hormone Receptor Status: Identification of patients at High Risk in Jableh- Syria. Research Journal of Pharmacy and Technology. 2018; 11(8): 3703-3710. doi:10.5958/0974-360X.2018.00680.7

6.      Al-Shalah MAN, Al-Mosawi HM, Alaawad AS. Comparative Study between Paired Primary and Relapsed Breast Cancer Patients based on Clinicopathological Features and Molecular Subtypes of Breast Cancer in Babylon Province. Research Journal of Pharmacy and Technology. 2018;11(6):2365-2371. doi:10.5958/0974-360X.2018.00439.0

7.      Al-Mahmood S, Sapiezynski J, Garbuzenko OB, Minko T. Metastatic and triple-negative breast cancer: challenges and treatment options. Drug Deliv Transl Res. 2018;8(5):1483-1507. doi:10.1007/s13346-018-0551-3

8.      Garrett JT, Arteaga CL. Resistance to HER2-directed antibodies and tyrosine kinase inhibitors. Cancer Biology & Therapy. 2011; 11(9): 793-800. doi:10.4161/cbt.11.9.15045

9.      Jernström S, Hongisto V, Leivonen S-K, et al. Drug-screening and genomic analyses of HER2-positive breast cancer cell lines reveal predictors for treatment response. Breast Cancer (Dove Med Press). 2017; 9: 185-198. doi:10.2147/BCTT.S115600

10.   Thalkari AB, Karwa PN, Zambare KK, Tour NS, Chopane PS. Paclitaxel Against Cancer: A new trademarked drug. Rese Jour of Pharmac and Phytoch. 2019; 11(3): 123. doi:10.5958/0975-4385.2019.00021.9

11.   Yao Y, Chu Y, Xu B, Hu Q, Song Q. Radiotherapy after surgery has significant survival benefits for patients with triple-negative breast cancer. Cancer Med. 2019;8(2):554-563. doi:10.1002/cam4.1954

12.   Swapnil K, Vijay S, Chandrakant M. Targeted Drug Delivery: A Backbone for Cancer Therapy. Asian Journal of Pharmaceutical Research. 2013; 3(1): 40-46.

13.   Sudhakar GK, Pai V, Pai A. An overview on current Strategies in Breast Cancer Therapy. Research Journal of Pharmacology and Pharmacodynamics. 2014; 5(6): 353-355.

14.   Saha D, Maity T, Jana M, Mandal S. Cancer Treatment Strategy-An Overview. Asian Journal of Pharmacy and Technology. 2011; 1(2): 28-33.

15.   Patidar A, S.C. Shivhare, Ateneriya U, Choudhary S. A Comprehensive Review on Breast Cancer. Asian Journal of Nursing Education and Research. 2012; 2(1): 28-32.

16.   Dange VN, Shid SJ, Magdum CS, Mohite SK. A Review on Breast cancer: An Overview. Asian Journal of Pharmaceutical Research. 2017; 7(1): 49-51. doi:10.5958/2231-5691.2017.00008.9

17.   Yadav AR, Mohite SK. Cancer- A Silent Killer: An Overview. Asian Journal of Pharmaceutical Research. 2020;10(3):213-216. doi:10.5958/2231-5691.2020.00036.2

18.   Damaskos C, Garmpi A, Nikolettos K, et al. Triple-Negative Breast Cancer: The Progress of Targeted Therapies and Future Tendencies. Anticancer Res. 2019;39(10):5285-5296. doi:10.21873/anticanres.13722

19.   Eldhose E, Gowramma B, Mohammed M, Kalirajan R, Kaviarasan L. Translational Chemotherapy for triple negative Breast Cancer - A Review on significance of poly (ADP-ribose) polymerase 1 (PARP 1) inhibitors. Research Journal of Pharmacy and Technology. 2019;12(6):3098-3104. doi: 10.5958/0974-360X.2019.00524.9

20.   McCann KE, Hurvitz SA, McAndrew N. Advances in Targeted Therapies for Triple-Negative Breast Cancer. Drugs. 2019;79(11):1217-1230. doi:10.1007/s40265-019-01155-4

21.   Abu Samaan TM, Samec M, Liskova A, Kubatka P, Büsselberg D. Paclitaxel’s Mechanistic and Clinical Effects on Breast Cancer. Biomolecules. 2019;9(12):789. doi:10.3390/biom9120789

22.   Wahba HA, El-Hadaad HA. Current approaches in treatment of triple-negative breast cancer. Cancer Biol Med. 2015;12(2):106-116. doi: 10.7497/j.issn.2095-3941.2015.0030

23.   Bardia A, Mayer IA, Vahdat LT, et al. Sacituzumab Govitecan-hziy in Refractory Metastatic Triple-Negative Breast Cancer. New England Journal of Medicine. 2019;380(8):741-751. doi:10.1056/NEJMoa1814213

24.   FDA U. FDA Approves New Therapy for Triple Negative Breast Cancer That Has Spread, Not Responded to Other Treatments. FDA. Published April 22, 2020. Accessed June 1, 2020. https://www.fda.gov/news-events/press-announcements/fda-approves-new-therapy-triple-negative-breast-cancer-has-spread-not-responded-other-treatments

25.   Meiyanto E, Putri H, Larasati YA, et al. Anti-Proliferative and Anti-Metastatic Potential of Curcumin Analogue, Pentagamavunon-1 (PGV-1), Toward Highly Metastatic Breast Cancer Cells in Correlation with ROS Generation. Advanced Pharmaceutical Bulletin. 2019; 9(3): 445-452. doi:10.15171/apb.2019.053

26.   Lestari B, Nakamae I, Yoneda-Kato N, et al. Pentagamavunon-1 (PGV-1) inhibits ROS metabolic enzymes and suppresses tumor cell growth by inducing M phase (prometaphase) arrest and cell senescence. Sci Rep. 2019; 9(1): 1-12. doi: 10.1038/s41598-019-51244-3

27.   Meiyanto E, Husnaa U, Kastian RF, et al. The Target Differences of Anti-Tumorigenesis Potential of Curcumin and Its Analogues Against HER-2 Positive and Triple-Negative Breast Cancer Cells. Advanced Pharmaceutical Bulletin. 2021;11(1):188-196. doi: 10.34172/apb.2021.020.

28.   Utomo RY, Wulandari F, Novitasari D, et al. Preparation and cytotoxic evaluation of PGV-1 derivative, CCA-1.1, as a new curcumin analog with improved-physicochemical and pharmacological properties. Advanced Pharmaceutical Bulletin, in Press.

29.   Ahlina FN, Nugraheni N, Salsabila IA, Haryanti S, Da’i M, Meiyanto E. Revealing the Reversal Effect of Galangal (Alpinia galanga L.) Extract Against Oxidative Stress in Metastatic Breast Cancer Cells and Normal Fibroblast Cells Intended as a Co- Chemotherapeutic and Anti-Ageing Agent. Asian Pacific Journal of Cancer Prevention. 2020;21(1):107-117. doi:10.31557/APJCP.2020.21.1.107

30.   Meiyanto E, Putri DDP, Susidarti RA, et al. Curcumin and its analogues (PGV-0 and PGV-1) enhance sensitivity of resistant MCF-7 cells to doxorubicin through inhibition of HER2 and NF-kB activation. Asian Pac J Cancer Prev. 2014;15(1):179-184.

31.   Hermawan A, Fitriasari A, Junedi S, et al. PGV-0 and PGV-1 increased apoptosis induction of doxorubicin on MCF-7 breast cancer cells. Pharmacon. 2011;12(2):55-59.

32.   Amalina ND, Nurhayati IP, Meiyanto E. Doxorubicin Induces Lamellipodia Formation and Cell Migration. Indonesian Journal of Cancer Chemoprevention. 2017; 8(2): 61–67.

33.   Liou G-Y, Storz P. Reactive oxygen species in cancer. Free Radic Res. 2010;44(5):479-496. doi:10.3109/10715761003667554

34.   Teppo H-R, Soini Y, Karihtala P. Reactive Oxygen Species-Mediated Mechanisms of Action of Targeted Cancer Therapy. Oxid Med Cell Longev. 2017; 2017: 1485283. doi:10.1155/2017/1485283

35.   Lee S, Lee J-S. Cellular senescence: a promising strategy for cancer therapy. BMB Rep. 2019;52(1):35-41. doi:10.5483/BMBRep.2019.52.1.294

36.   Plesca D, Mazumder S, Almasan A. DNA Damage Response and Apoptosis. Methods Enzymol. 2008; 446: 107-122. doi:10.1016/S0076-6879(08)01606-6

37.   Castedo M, Coquelle A, Vivet S, et al. Apoptosis regulation in tetraploid cancer cells. The EMBO Journal. 2006;25(11):2584-2595. doi: 10.1038/sj.emboj.7601127

38.   Fragkos M, Beard P. Mitotic Catastrophe Occurs in the Absence of Apoptosis in p53-Null Cells with a Defective G1 Checkpoint. PLOS ONE. 2011;6(8): e22946. doi:10.1371/journal.pone.0022946

39.   Gee M, Margaret M. Targeting the Mitotic Catastrophe Signaling Pathway in Cancer. Mediators of Inflammation. doi:https://doi.org/10.1155/2015/146282

40.   Tanaka K, Goto H, Nishimura Y, Kasahara K, Mizoguchi A, Inagaki M. Tetraploidy in cancer and its possible link to aging. Cancer Sci. 2018;109(9):2632-2640. doi:10.1111/cas.13717

41.   Eom Y-W, Kim MA, Park SS, et al. Two distinct modes of cell death induced by doxorubicin: apoptosis and cell death through mitotic catastrophe accompanied by senescence-like phenotype. Oncogene. 2005;24(30):4765-4777. doi: 10.1038/sj.onc.1208627

42.   Lee T, Lau T, Ng I. Doxorubicin-induced apoptosis and chemosensitivity in hepatoma cell lines. Cancer Chemother Pharmacol. 2002;49(1):78-86. doi:10.1007/s00280-001-0376-4

43.   Hung J-Y, Wen C-W, Hsu Y-L, et al. Subamolide A Induces Mitotic Catastrophe Accompanied by Apoptosis in Human Lung Cancer Cells. Evidence-Based Complementary and Alternative Medicine. doi:https://doi.org/10.1155/2013/828143

44.   Masgras I, Carrera S, Verdier PJ de, et al. Reactive Oxygen Species and Mitochondrial Sensitivity to Oxidative Stress Determine Induction of Cancer Cell Death by p21. J Biol Chem. 2012;287(13):9845-9854. doi:10.1074/jbc.M111.250357

45.   Li H, Hu P, Wang Z, et al. Mitotic catastrophe and p53-dependent senescence induction in T-cell malignancies exposed to nonlethal dosage of GL-V9. Arch Toxicol. 2020;94(1):305-323. doi:10.1007/s00204-019-02623-2

46.   Vitale I, Galluzzi L, Castedo M, Kroemer G. Mitotic catastrophe: a mechanism for avoiding genomic instability. Nature Reviews Molecular Cell Biology. 2011;12(6):385-392. doi:10.1038/nrm3115

47.   Bharadwaj D, Mandal M. Senescence in polyploid giant cancer cells: A road that leads to chemoresistance. Cytokine & Growth Factor Reviews. 2020;52:68-75. doi:10.1016/j.cytogfr.2019.11.002

48.   Wang Q, Wu PC, Dong DZ, et al. Polyploidy road to therapy-induced cellular senescence and escape. International Journal of Cancer. 2013;132(7):1505-1515. doi:10.1002/ijc.27810

49.   Bielak-Zmijewska A, Wnuk M, Przybylska D, et al. A comparison of replicative senescence and doxorubicin-induced premature senescence of vascular smooth muscle cells isolated from human aorta. Biogerontology. 2014;15(1):47-64. doi:10.1007/s10522-013-9477-9

50.   Beauséjour CM, Krtolica A, Galimi F, et al. Reversal of human cellular senescence: roles of the p53 and p16 pathways. EMBO J. 2003;22(16):4212-4222. doi:10.1093/emboj/cdg417

51.   Elmore LW, Rehder CW, Di X, et al. Adriamycin-induced senescence in breast tumor cells involves functional p53 and telomere dysfunction. J Biol Chem. 2002;277(38):35509-35515. doi:10.1074/jbc.M205477200

52.   Yerlikaya A, Okur E, Ulukaya E. The p53-independent induction of apoptosis in breast cancer cells in response to proteasome inhibitor bortezomib. Tumour Biol. 2012;33(5):1385-1392. doi:10.1007/s13277-012-0386-3

53.   Marinello J, Delcuratolo M, Capranico G. Anthracyclines as Topoisomerase II Poisons: From Early Studies to New Perspectives. Int J Mol Sci. 2018;19(11). doi:10.3390/ijms19113480

 

 

 

Received on 29.06.2020           Modified on 16.10.2020

Accepted on 26.11.2020         © RJPT All right reserved