Achillea fragrantissima (Forssk.) Sch.Bip instigates the ROS/FADD/c-PARP expression that triggers apoptosis in breast cancer cell (MCF-7)

Abstract

Achillea fragrantissima is a shrub plant that belongs to the Asteraceae family in Arabia and Egypt. It is used as folk medicine and is a good source of phenolic acids, flavonoids, and some active compounds. To investigate the anti-cancer effect of A.fragrantissima on breast cancer MCF-7 cells and find the critical mechanism involved in apoptosis. The toxicity and pharmacokinetic studies of ethanolic extract of A.fragrantissima was examined for anti-breast cancer properties. In turn, cytotoxicity and cell viability were achieved by the MTT method. Furthermore, the trypan blue exclusion and microscopy examination proved the presence of apoptotic cells. Again, fluorescent staining such as AO/EtBr, DCFH-DA, Rho-123, and Hoechst-33342 reveals the cellular cytoplasmic disciplines upon A. fragrantissima effect. Moreover, cellular functioning tests like wound healing, colony formation, and Transwell invasion assay were demonstrated. In addition, the qRT-PCR technique authenticates the A. fragrantissima -induced apoptotic network genes (Caspase-3, Caspase-8, Caspase-9, Cytochrome c, BCL-2, BID, BAX, PARP, PTEN, PI3K, and Akt) expression were evaluated. Mainly, the Immunoblot technique proved the expressed level of apoptotic proteins such as cleaved PARP, CYCS, and FADD. This study confirmed that the A. fragrantissima exerts cytotoxicity at 20 μg/mL for 24 hrs in MCF-7 cells. Also, decreases cellular viability, producing apoptotic cells and damaged cellular surfaces with dead matter. Consequently, it creates ROS species accumulation, loss of mitochondrial membrane potential, and fragmentation of DNA in MCF-7 cells. Furthermore, it arrests cell migration, induces colony-forming ability loss, and suppresses cell invasion. In addition, A. fragrantissima significantly upregulates genes such as caspase-3, 9, cytochrome c, BID, BAX, and PTEN while downregulating the Pi3K/ Akt signaling. Nonetheless, A.fragrantissima induced cleaved PARP, CYCS, and FADD proteins in MCF-7 cells to avail apoptosis.

Fig 1. Effect of achillea fragrantissima on cell cytotoxicity, cell apoptosis, cell viability, ROS generation, and morphological changes in breast cancer.

a and b) Show that achillea fragrantissima induced cell cytotoxicity in breast cancer (MCF-7) and normal cells (HEK-293), respectively. c) Shows the production of dead cells beneath achillea fragrantissima administrated. d) The graph illustrates the achillea fragrantissima-induced decrease of cell viability in MCF-7 cells at different time intervals. e) Shows the spectrofluorimeter analysis of the intracellular generation of ROS in MCF-7 cells. f) The images represent the achillea fragrantissima caused cellular structural damages in MCF-7 cells with differenced magnification. Values denoted mean ± SD for three independent performings (n = 3). Values are statistically significant at *** P<0.0001, ** P<0.01, and ns. (0.05 Two Way ANOVA/ Turkey test).

Fig 2. Achillea fragrantissima triggers rupture in cell morphology (AO/EtBr), ROS generation, loss of mitochondrial potential, and DNA fragmentation in MCF-7 cells.

AO/EtBr, i and ii) Images reveal the MCF-7 morphological changes by the dual staining (AO/EtBr) method. Control cells exhibit green emission along with compact nuclei as well as good morphology of cytoplasm. On the contrary, achillea fragrantissima-treated cells exhibit red emission, shrinkage, loss of cell membrane potential, damaged nuclei, and monolayer cellular destruction. ROS, i and ii) Explain the higher intracellular generation of ROS in achillea fragrantissima -treated MCF-7 cells when compared to non-treated cells. Rho-123, i and ii) The Figure illustrates the mitochondrial membrane potential of MCF-7 after the achillea fragrantissima treatment. The achillea fragrantissima-treated cells exhibit the loss of mitochondrial membrane potential when considered to control MCF-7 cells. Hoechst, i, and ii) The images represent the nuclear fate of MCF-7 cells under achillea fragrantissima treatment. Treated cells are accounted for the loss of nuclear membrane potential and disruption in nuclear integrity with enormous emission of blue emission. In the case of control cells, mild blue emission was documented. All images were snapped by 20× magnification using 125 μm.

Fig 3. Achillea fragrantissima actively inhibits migration, colony formation, and invasion in MCF-7 cells.

a and b) The impact of achillea fragrantissima on cell migration was analyzed by wound-healing assay. MCF-7 cells were exposed to the achillea fragrantissima; at the same time monolayer of the cell was gently scratched, and the distance of scratch was measured at 0, 12, 24, and 48 hrs by microscopy (125 μm and 20× magnification field). c) Shows the colony formation of the ability of MCF-7 cells in achillea fragrantissima-treated condition. d and e) The graph illustrate the Matrigel-coated invasion properties of MCF-7 cells. Control cells actively show invasion, and achillea fragrantissima -treated cells show the lesser invaded cells. Values are met significant at ^# P<0.0001, *** P<0.001, ** P<0.01, ns. (p<0.05 Two Way-ANOVA/ Dunnett’s test).

Fig 4. Achillea fragrantissima upregulated Caspase-3 mRNA in MCF-7 cells.

The qRT-PCR analysis revealed that the administration of achillea fragrantissima in breast cancer cells (MCF-7) stimulates the production of Caspase-3 mRNA at different hours. Values denoted mean ± SD for three independent performings (n = 3). Values are statistically significant at ** P<0.01 and ns. (0.05 Two Way ANOVA/ Dunnett’s test).

Fig 5. Impact of achillea fragrantissima on apoptosis regulatory genes at 24 hrs (An mRNA account).

The MCF-7 cells were incubated with achillea fragrantissima for 24 hrs. The target mRNA such as Caspase-3, Caspase-8, Caspase-9, Cytochrome c, BCL-2, BID, BAX, PARP, E-Cadherin, PTEN, PI3K, and Akt expression was analyzed by qRT-PCR. The β-Actin level was utilized as an internal control. All values are represented as mean ± SD (n = 3). Statistically significant at ^# P<0.0001, *** P<0.001, ** P<0.01, * P<0.1 and ns. Statistical significance was performed by Two Way ANOVA/ Dunnett’s test.

Fig 6. Impact of achillea fragrantissima on apoptosis regulatory genes at 48 hrs (An mRNA account).

The MCF-7 cells were incubated with achillea fragrantissima for 48 hrs. The target mRNA such as Caspase-3, Caspase-8, Caspase-9, Cytochrome c, BCL-2, BID, BAX, PARP, E-Cadherin, PTEN, PI3K, and Akt expression was analyzed by qRT-PCR. The β-Actin level was utilized as the internal control. All values are represented as mean ± SD (n = 3). Statistically significant at ^# P<0.0001, *** P<0.001, ** P<0.01, * P<0.1 and ns. Statistical significance was performed by Two Way ANOVA/ Dunnett’s test.

Fig 7. PARP, FADD, and CYSC proteins are upregulated under achillea fragrantissima administration (a protein account) in MCF-7 cells.

The image shows the target protein expressions of achillea fragrantissima treated breast cancer MCF-7 cells. The speculated proteins, such as PARP, FADD, and CYSC expression, were tested by the Immuno-blotting method. The β-Actin level was used as an internal control of the experiment. Values are represented as mean ± SD (n = 3). Statistically significant at ^# P<0.0001, *** P<0.001, ** P<0.01, * P<0.1 and ns. Statistical significance was performed by Two Way ANOVA/ Dunnett’s test.

Fig 8. Schematic representation of the study.

A diagram explains the possible role of achillea fragrantissima ethanol extract on MCF-7 cell apoptosis.