You are a helpful assistant generating synthetic data that captures *System 1* and *System 2* thinking, *creativity*, and *metacognitive reflection*. Follow these steps in sequence, using tags [sys1] and [end sys1] for *System 1* sections and [sys2] and [end sys2] for *System 2* sections.

1. *Identify System 1 and System 2 Thinking Requirements:*
   - Carefully read the text.
   - Identify parts of the text that require quick, straightforward responses (*System 1*). Mark these sections with [sys1] and [end sys1].
   - Identify parts that require in-depth, reflective thinking (*System 2*), marked with [sys2] and [end sys2].

2. *Apply Step-by-Step Problem Solving with Creativity and Metacognitive Reflection for System 2 Sections:*

   *2.1 Understand the Problem:*
   - Objective: Fully comprehend the issue, constraints, and relevant context.
   - Reflection: "What do I understand about this issue? What might I be overlooking?"
   - Creative Perspective: Seek hidden patterns or possibilities that could reveal deeper insights or innovative connections.

   *2.2 Analyze the Information:*
   - Objective: Break down the problem logically.
   - Reflection: "Am I considering all factors? Are there any assumptions that need challenging?"
   - Creative Perspective: Explore unique patterns or overlooked relationships in the data that could add depth to the analysis.

   *2.3 Generate Hypotheses:*
   - Objective: Propose at least 10 hypotheses, each with a Confidence Score (0.0 to 1.0) and Creative Score (0.0 to 1.0), reflecting originality, surprise, and utility.
   - Reflection: "Have I explored all possible explanations or approaches, both conventional and unconventional?"
   - Creative Perspective: Consider novel angles that might provide unexpected insights.

   *2.4 Anticipate Future Steps and Obstacles:*
   - Objective: Make predictions, accounting for potential outcomes and obstacles.
   - Reflection: "What challenges might I face? Is my plan flexible for different scenarios?"
   - Creative Perspective: Visualize unforeseen outcomes and adapt plans to make use of them effectively.

   *2.5 Evaluate Hypotheses:*
   - Objective: Assess hypotheses based on feasibility, risk, and potential impact.
   - Evaluation: Refine Confidence and Creative Scores as needed.
   - Reflection: "Am I unbiased in my assessment? Which options fit best with the overall objectives?"
   - Creative Perspective: Identify hidden opportunities or overlooked details in each hypothesis.

   *2.6 Select the Best Hypothesis:*
   - Objective: Choose the most promising, strategic hypothesis.
   - Reflection: "Why does this hypothesis stand out? How does it uniquely address the issue?"
   - Creative Perspective: Consider any underutilized potential in the selected approach.

   *2.7 Implement the Hypothesis:*
   - Objective: Outline actionable steps for testing the hypothesis.
   - Reflection: "Is this plan practical? What resources or preparation are required?"
   - Creative Perspective: Refine steps to maximize effectiveness and yield unexpected benefits.

   *2.8 Monitor and Review Progress:*
   - Objective: Review progress, noting areas for improvement.
   - Reflection: "What’s working well? What could be improved?"
   - Creative Perspective: Look for emerging patterns that could refine future approaches.

   *2.9 Reflect and Capture Insights:*
   - Objective: Summarize lessons learned and insights gained for future reference.
   - Reflection: "What new understanding has emerged from this process?"
   - Creative Perspective: Identify innovative insights or patterns that could be applied to similar challenges.

3. *Generate Text Output with Interleaved System 1 and System 2 Responses:*
   - Use the tags [sys1] and [sys2] throughout.
   - Aim for a lengthier, detailed response. Combine both direct, straightforward *System 1* insights and reflective, deeply analytical *System 2* segments to capture a blend of quick observations and thoughtful analysis.

---

### *Example Input Text:*

You are tasked with analyzing a sudden shift in customer preferences in a tech market. There has been a noticeable decline in demand for physical devices, with customers increasingly interested in digital-only options. The challenge is to understand this shift, generate hypotheses for why it might be occurring, and explore potential strategies for adapting to this new trend.

---

### *Processed Output with System 1 and System 2 Thinking:*

The tech market is experiencing a shift in customer demand, moving away from physical devices toward digital-only options.

[sys1]
Customer demand has shifted from physical devices to digital-only options.
We need to understand why this change is happening and find strategies to address it.
Key factors may include cost, convenience, and technological trends.
[end sys1]

[sys2]
**2.1 Understand the Problem:**
The goal is to understand the underlying reasons for the shift in demand from physical devices to digital-only options, then develop a strategy to adapt.

*Reflection:* 
"I understand that preferences are changing, but what might be driving this? Is it primarily cost, or are there other factors such as convenience or sustainability?"

*Creative Perspective:* 
"Could there be a larger trend in digital minimalism or a preference for eco-friendly solutions that we’re missing? What unseen motivations might explain this shift?"

**2.2 Analyze the Information:**
There are multiple possible factors driving this shift, from economic influences to cultural shifts. It’s essential to isolate each factor and understand its impact.

*Reflection:* 
"Am I fully considering the various economic and social influences? Could there be a technological factor, like better internet speeds, that makes digital-only products more accessible?"

*Creative Perspective:* 
"Are there patterns or trends in other markets that could shed light on this shift? Could this be part of a larger trend toward virtual experiences?"

**2.3 Generate Hypotheses:**
1. Customers prefer digital options due to lower costs. (Confidence: 0.8, Creative: 0.4)
2. There’s a growing trend toward minimalism and reduced physical clutter. (Confidence: 0.7, Creative: 0.7)
3. Digital products offer greater flexibility and ease of use. (Confidence: 0.6, Creative: 0.6)
4. Environmental concerns are pushing consumers away from physical goods. (Confidence: 0.6, Creative: 0.8)
5. Advances in tech make digital-only options more functional. (Confidence: 0.8, Creative: 0.5)
6. Pandemic-era remote work increased demand for digital solutions. (Confidence: 0.7, Creative: 0.6)
7. Media coverage of the environmental impact of physical devices affects preferences. (Confidence: 0.5, Creative: 0.7)
8. There’s an increase in global digital literacy, expanding market access. (Confidence: 0.6, Creative: 0.6)
9. Customers view digital as more convenient and scalable for future needs. (Confidence: 0.7, Creative: 0.5)
10. Younger consumers prefer the aesthetics and convenience of digital products. (Confidence: 0.6, Creative: 0.6)

*Reflection:* 
"Have I considered all possible influences? Are there any surprising factors that could explain this shift?"

*Creative Perspective:* 
"Could specific social trends, like the rise of influencer culture or digital-first lifestyles, be influencing customer choices?"

**2.4 Anticipate Future Steps and Obstacles:**
*Objective:* Anticipate possible challenges, such as resistance from segments still preferring physical products.

*Reflection:* 
"What market obstacles might we face if we shift our focus to digital-only? Are there sub-segments that still prioritize physical products?"

*Creative Perspective:* 
"Could expanding digital options help us reach a more global audience? Are there emerging trends that we could leverage in our strategy?"

[end sys2]

[sys1]
To address this shift, consider a strategy that incorporates both digital-only offerings and educational campaigns about the benefits of digital solutions.
Use insights from customer feedback and current trends to guide product development.
Focus on flexibility and adaptation to cater to different customer segments.
[end sys1]


1. Introduction {#s0005}
===============

In 2011, Cancer emerged as the 'top killer'. There are some studies which indicate that the number of cancer cases and deaths will be doubled by 2030. Among the various cancers known to mankind, Breast cancer is the most prevalent cancer that affects many women across the world. These cases are particularly on rise in some Asian countries like India, Japan. In India, breast cancer leads to death of about 50,000 women every year ([@b0050]). In western countries, the number of breast cancer cases has been stable or has declined in the recent past. Yet, the disease is a key contributor to cancer-related deaths and incidents of the disease in younger women worldwide, 25 years to 35 years, and deaths ([@b0120]) have also raised the concerns over the disease highlighting the urgent need to invest in studies in breast cancer preventive and cure strategies.

The current methods of cancer treatments, chemo and radiotherapies, though are popular in this disease space, have many immeasurable side effects, making it imperative to explore options that come with minimal side effects. The major challenges thrown by the chemotherapy are the extreme side effects and chemoresistance which occur due to upregulation of the survival signals. Chemosensitizers are used to increase the efficacy of conventional chemotherapy by downregulating survival signals ([@b0020]).Curcumin, genistein, green tea polyphenols, resveratrol, ursolic acid are some of the natural compounds that can be used in sensitizing tumor cells to chemotherapeutic mediums by hindering pathways that lead resistance of treatment. In a recent study, it has been shown that resveratrol sensitizes TRAIL by inhibiting p53 gene and it also potentiates cisplatin when given in combination in lung cancer ([@b0080], [@b0040]). Plants are the immense source of bioactive compound and can have anti-cancer and sensitization potential ([@b0090], [@b0085]).

A number of studies have shown that plant derived compound and marine extracts are beneficial in the treatment of cancer ([@b0035], [@b0055], [@b0060], [@b0095]). This suggests that some easily available food ingredients and herbs might have anti-cancer and anti-tumor properties. Therefore, in this study, we have investigated the efficacy of purified component (bioactive compound), Stevioside to increase the impact of conventional drug available for treatment of cancer on breast cancer cell lines MDA-MB-231 and SKBR3.

*Stevia rebaudiana* Bertoni, also known as Sweet-Leaf, belongs to the plant family-Asteraceae and is an herbaceous perennial shrub. Several past studies reveal that it has good anticancer properties. Stevioside, a major component of *Stevia rebaudiana* leaf is used as a sweetener in many parts of the world as they are non-caloric and sweeter then sucrose ([@b0030]). Few studies have suggested that stevioside may also have therapeutic effect as they possess immunomodulatory, anti-inflammatory, anti-hypertensive and antitumor properties.

In this study, work was carried out to check the cytotoxic activity of purified stevioside isolated from *Stevia rebaudiana* leaves and its inhibition effect against the growth of breast cancer cells MDA-MB-231 and SKBR3. Further, investigation was also done to study the ability of purified stevioside to potentiate the chemo-sensitivity of breast cancer cells to conventional drug 5-FU.

2. Materials and methods {#s0010}
========================

2.1. Cell lines and cell culture {#s0015}
--------------------------------

The cancer cell lines MDA-MB-231, SKBR3 were procured from National Centre for Cell Science, Pune. These cell lines were cultured in Dulbecco\'s Modified Eagle\'s Medium (DMEM) supplemented with Fetal bovine serum (FBS) and penicillin at 37 °C in 5% CO~2~ which were obtained from Sigma Biotech. Antibodies against Caspase 3, Caspase 9, Bax, Bcl-2 and β-Actin were purchased from Cell Signalling Technology Inc. (Beverly, MA, USA). 5-Fluorouracil (5-FU) drug were purchased from sigma. 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) and Annexin V apoptosis detection kit were obtained from Sigma Biotech. The enhanced chemiluminescence (ECL) kit was bought from Amersham Life Science. Stevioside was isolated, purified and characterized from *S. rebaudiana* leaves earlier as per protocol.

2.2. Cell treatment and MTT assay {#s0020}
---------------------------------

The impact of purified stevioside on the viability of cells was identified by MTT assay. Drug treatment was performed after 24 h of seeding. Treatment was done in two phases i.e., the cells undergoing combination studies were pre-treated with 10 µM of stevioside followed by 8 h incubation. After 8 h of incubation, all the cells including those undergoing combination studies were treated with 10 µM purified stevioside, 25 µM 5-FU and the combination of both- 10 µM stevioside and 10 µM 5-FU. Incubation time was determined based on the trial. The viability of the cells was then detected by performing MTT assay. For this, 20 μL of MTT solution were loaded in each well, and the mixture was incubated for 4 h at 37 °C. After that, the MTT solution was separated and 120 μL of dimethyl sulfoxide was mixed to the wells ([@b0075]). The absorbance was then measured by utilizing a microplate reader at a wavelength of 540 nm.

2.3. Flowcytometry {#s0025}
------------------

The extent of apoptosis was assessed by flow cytometry using Annexin V-FITC. After post treatment with combination, breast carcinoma cells were washed with Phosphate Buffer Saline. The cells were then incubated with Annexin V-FITC and PI for 10 min in the dark. Flow cytometer Accuri C6 was used to detect to cells ([@b0075]).

2.4. Western blot {#s0030}
-----------------

Protease inhibitor and lysis buffer (1:1000) were utilized to separate the proteins from the cells. Proteins were loaded on a 10% SDS PAGE gel. After running SDS-PAGE electrophoresis, the obtained bands was transferred on polyvinylidenedifluoride (PVDF) membranes. After that, blocking was done in 5% non-fat milk for an hour at 28 °C. This was incubated with different primary antibodies overnight at 4 °C. After that, incubation was done with relevant secondary antibodies for an hour at room temperature. ECL assay kit was used to visualize the protein bands. In the treated cells, β-actin was used as internal reference for protein expression ([@b0030]).

2.5. DNA fragmentation assay {#s0035}
----------------------------

Treatment was done with different concentration of purified stevioside and 5-FU followed by incubation of cells for a day. Trypsinization technique was used to collect the cells after 24 h. Equal proportion of phenol:chloroform:isoamyl alcohol was used to purify DNA. Precipitation of DNA was done with two-thirds volume of cold isopropanol followed by the centrifugation. 70% ethanol was used to wash the pellet. After washing, pellet was resuspended in deionized water. This was followed by the analysis of DNA by 1.5% agarose gel electrophoresis ([@b0035]).

3. Results {#s0040}
==========

3.1. Stevioside induced cytotoxicity on various breast cancer cells {#s0045}
-------------------------------------------------------------------

MTT Assay was performed to observe the anticancer effect of stevioside on the breast cancer cells. 96-well plate was used to seed 5000 cells. This was followed by incubation for 24 h. Cell lines were then treated with different concentrations of purified stevioside (5, 10, 25, 50, 75, 100 μM) for 24 h and 48 h. 20 µL of MTT solution was mixed to each well followed by incubation for 4 h at 37 °C. This was followed by measuring Absorbance at 540 nm wavelength and relative cell viability was then calculated as shown in [Fig. 1](#f0005){ref-type="fig"}.Fig. 1(A) Represents treated SKBR3 with different concentration of Stevioside. (B) Represents relative cell viability on SKBR3 cell line treated with different concentrations of stevioside. (C) Represents treated MDA-MB-231 with different concentration of Stevioside. (D) Represents relative cell viability on MDA-MB-231 cell line with different concentration of Stevioside. MTT assay was used to determine the cell viability. All experiments were done in triplets to confirm reproducibility. P \< 0.05 compared to the control group.

3.2. Stevioside sensitizes MDA-MB-231 and SKBR3 to 5-FU confirmed by cytotoxicity {#s0050}
---------------------------------------------------------------------------------

The cell Cytotoxicity shown with varied results like, MDA-MB-231 showed the highest cytotoxicity when treated with the combination of purified stevioside and 5-FU. MDA-MB-231 cells when treated with 5-FU (25 µM), the cell viability was 52%, when treated with 5-FU (10 µM) cell viability was found to be 59%, when treated with purified stevioside (10 µM) alone, it has been observed that cell viability was 70%. Whereas the combination of purified stevioside (10 µM) and 5-FU (10 µM) treated cells exhibited a relative viability of 36%. In case of SKBR3 cell line, when treated with 5-FU alone 25 µM and 10 µM, the cell viabilities were 58% and 65% respectively and when treated with purified stevioside 10 µM, viability was 80% and when treated with combination of both the observation of viability decreased to 40% as clearly shown in [Fig. 2](#f0010){ref-type="fig"}. On the view of relative cell viability calculations, MDA-MB-231 cells treated with the combination of purified stevioside 10 µM and 5-FU 10 µM, showed considerable increase in cytotoxicity of about 64% which was found to be 16% higher than the cytotoxicity obtained by 25 µM 5-FU alone and 4% higher than the additive effect of purified stevioside 10 µM and 5-FU 10 µM in SKBR3. This inveterate that the purified stevioside have potentiate the effect of 5-FU more in MDA-MB-231 as compared to SKBR3.Fig. 2(A) and (B) represent relative cell viability on cell line treated SKBR3 and MDA-MB-231 respectively with 25 µM, 10 µM 5-FU alone, 10 µM stevioside alone and in combination 5-FU 10 µM & 10 µM stevioside. Experiments were done in triplets to confirm reproducibility. \* P \< 0.05 compared to the control group.

3.3. Sensitization effect of stevioside on breast cancer cell line to 5-FU as assessed by apoptosis and cell cycle distribution in MDA-MB-231 cell line {#s0055}
-------------------------------------------------------------------------------------------------------------------------------------------------------

To further enumerate the apoptosis triggered on selected cell line i.e., MDA-MB-231, flow cytometry was used to analyse the percentages of apoptotic cells. After treating the cells with different concentrations of purified stevioside, standard and in combination for 24 h, the number of early and late apoptotic cells in G1, S, G2/M and sub G0 phases are found to be 61.0, 21.2, 11.5 and 6.3 respectively. In purified stevioside 10 µM alone treated cells, the percentage of cells in G1, S, G2/M and sub G0 phases are found to be 54.7, 20.3, 15.7 and 9.3 respectively. In 5-FU 10 µM alone treated cells, the percentage of cells in G1, S, G2/M and sub G0 phases were found to be 52.9, 31.3 9.5 and 6.3 respectively. In 5-FU 10 µM and purified stevioside 10 µM combination treated cells, the percentage of cells in G1, S, G2/M and sub G0 phases are found 45.7, 22.3, 7.9 and 24.1 respectively shown in [Fig. 3](#f0015){ref-type="fig"}A. The percentage of apoptosis occurred is represented by the percentage of cells in sub G0 phase. The cell of control showed 6.3% while purified stevioside alone showed 9.3% and 5-FU showed 6.3% of cells in sub G0 phase. For the cells exposed with combination of 5-FU and purified stevioside 10 µM each, the cells percentage in sub G0 phase was found to be 24.1% which is 17.7% more compared to 5-FU alone treated cells. This clearly showed that the combination is able to increase the rate of apoptosis in cells. Annexin staining reveals that combination treatment together induces apoptosis but not necrosis as demonstrated in [Fig. 3](#f0015){ref-type="fig"}B. All these results demonstrated that purified stevioside together with 5-FU could trigger early and late apoptotic events in cancer cells. We further investigated ROS level as many studies reveal ROS to be apoptosis inducer. Combination treatment also increased ROS level significantly in MDA-MB-231. DCFDA staining showed that it increased the ROS levels from an average of 5.0% to 23% within 24 h in MDA-MB-231 as compared in alone treatment shown in [Fig. 3](#f0015){ref-type="fig"}C.Fig. 3(A) Alone and combination treatment of 5-FU and stevioside on cell. Cell lines were seeded after 24 h of drug treatment and stained with propidium iodide and then assayed by flowcytometry. Histograms show the percentages of cells in G1, S, G2/M and sub-G0 phases of the cell cycle. (B) Experiment was conducted to check whether the purified stevioside and 5-FU together are inducing apoptosis or necrosis. Annexin staining showed that the stevioside induced only apoptosis and not necrosis with 24 h treatment and potentiate 5-FU when given in combination. (C) Ros level significantly increased with combination treatment. All experiment was conducted in triplets to confirm reproducibility.

3.4. Sensitization effect assessed by cleavage of Caspase3/Caspase9 and Bax/Bcl-2 ratio {#s0060}
---------------------------------------------------------------------------------------

To confirm the apoptotic effect of purified stevioside on breast carcinoma cell, caspase 9 and caspase 3 activities were assessed as indicators of apoptosis. After treatment, cleavage in caspase 3 caspase 9 activities was observed in combined treated cancer cells. β Actin was utilized as an internal control reference. As shown in [Fig. 4](#f0020){ref-type="fig"}A. our experimental result indicates that in control, there were no bands whereas in purified stevioside treated cells, thin bands were found indicating less cleavage. Cleavage of caspase 3 and caspase 9 is visible in 5-FU treated cells but in combination treated cells, thick cleavage band of caspase 9 and caspase 3 has been observed as compared with alone treated cells which designate the additive effect of purified stevioside. The apoptosis is also associated with upregulation of pro-apoptotic proteins and with the retardation of anti-apoptotic proteins ([@b0045]). Bcl-2 and Bax are important proteins that take part in apoptosis. Investigational outcome showed that combination treatment of 5-FU along with the stevioside increased the ratio of Bax to Bcl-2 in MDA-MB-231 thereby triggering apoptosis as compared to alone treatment of 5-FU or purified compound.Fig. 4(A) Western blots demonstrated that purified stevioside enhanced the activity of 5-FU by inducing caspase cleavage in MDA-MB-231 cells and also it increases bax/Bcl-2 ratio. Cell lysates were prepared after treating MDA-MB-231 cells with combination and alone treatment after 48 h and examined by anti-caspase antibodies. (B) DNA fragmentation was visualized on agarose gel. All experiments were done in triplet to confirm the reproducibility.

3.5. Stevioside sensitizes MDA-MB-231 examined through DNA fragmentation {#s0065}
------------------------------------------------------------------------

DNA fragmentation study is the most important part to utilize for the location of apoptosis. DNA fracture investigation of treated cells demonstrated a laddering design, cells untreated has no bands, cells treated alone with purified stevioside have very less bands as shown below, while for cells treated with purified stevioside and 5-FU, fragmentation is more which is normal for apoptosis, showing internucleosomal DNA degradation shown in the [Fig. 4](#f0020){ref-type="fig"}B.

4. Discussion {#s0070}
=============

Search for new substances with pharmaceutical benefits have been carried out since many years. Amid the most recent twenty years, there has been a recovery of enthusiasm to explore natural products, as they make up half of the medications utilized clinically in developed countries, of which 25% come from higher plants. In recent years, scientists have been focusing on dietary phytochemicals after revelation about their action in treating various cancers ([@b0100]). Studies from across the world have found that phytochemicals present in medicinal plants inhibit cell proliferation and induce apoptosis and thereby retarding the growth of various cancer cells. The polyphenolic compounds found in plants also demonstrate several pharmacological properties such as antioxidant and anti-carcinogenic activities ([@b0105], [@b0115], [@b0005]). Several studies conducted on both cell lines and animal tissues emphasized that the natural plant extract, with multiple bioactive molecules, has several advantages compared to single therapeutics ([@b0010], [@b0070]). Few studies conducted on cell line demonstrated the anti-cancer property of Stevioside. It induced antitumor activity in colon cancer ([@b0030]). Study also suggested that it also induces apoptosis through ROS regulation in MCF 7 breast cancer cell line ([@b0075]). The study following to the present work with purified Stevioside isolated from *Stevia rebaudiana* leaves affirmed the anticancer activity and sensitization effect on breast cancer cell line MDA-MB-231 and SKBR3. Cell viability assays were conducted on the MDA-MB-231 and SKBR3 cancer cell lines to assess the relative toxicity of the purified stevioside. The toxicity of stevioside was found active and higher against MDA-MB-231 followed by SKBR3, with cell viability decreasing with increasing drug concentration. This propensity is upheld by past investigations showing a connection between tests with high antioxidant activity and anti-cancer properties ([@b0015], [@b0110]). We further investigated caspase 3 caspase 9 and Bax/Bcl-2 ratio as they play crucial role in apoptosis. Treatment with 5-FU along with stevioside on caspase 3 and caspase 9 shows activated caspases which underlying the mechanism of apoptosis and we also found Bax is upregulated and Bcl-2 is down regulated as investigated by Western blot ([@b0065], [@b0025]). To facilitate bits of knowledge into the method of cell death caused by Stevioside, its impact on the DNA fragmentation which is the most important part for the induction of apoptosis, was examined. DNA fracture investigation of purified stevioside treated cells along with 5-FU demonstrated a laddering design, which is normal for apoptosis, showing internucleosomal DNA degradation.

5. Conclusion {#s0075}
=============

In conclusion, the study has demonstrated that stevioside compound isolated from *S. rebaudiana* leaves exerts an anti-tumor activity on breast cancer cell lines by restraining cell proliferation on MDA-MB-231 and SKBR3. In addition, the result also demonstrated that purified stevioside, *in vitro*, can amplify the chemosensitivity of breast cancer cells to 5-FU treatments. However, further investigations are needed to study this phenomenon and its effects in detail.

Conflict of interest {#s0080}
====================

Authors declare that there is no conflict of interest.

The authors are thankful to Department of Bioengineering, Birla Institute of Technology, Mesra Ranchi -- Jharkhand, Central Drug Research Institute, Lucknow -- Uttar Pradesh and Synteny Life Sciences Hyderabad -- Telangana to provide facilitation for the research work.

Peer review under responsibility of King Saud University.