Intracellular, Extracellular, and Cytotoxic Effects Of Abrus Agglutinin On Tumor Cells: A Comprehensive Review

Abstract

Half of all people will develop cancer during their lifetime. That makes it vitally important to discover new, less toxic ways to treat cancer. The plant molecule Abrus agglutinin (AGG) is a promising anti-tumor molecule that has been shown to increase pro-apoptotic—or pro-death—biomolecules and decrease proliferative ones in multiple different cancer cell lines. Another main benefit to AGG is its selectiveness. In mice, AGG did not show any toxic side effects at 1mg/kg and below, and at this dose, AGG still successfully targeted cancerous cells without harming normal cells. This review confirms the potential of AGG as a chemotherapeutic agent through intracellular and extracellular methods and selective cytotoxicity.

Introduction

Cancer is the second leading cause of death in the United States [1] and treatment can come with significant long and short-term side effects. This has led many researchers to look for safer alternatives to common chemotherapeutic agents, as these therapies lack specificity toward cancer cells, resulting in harm to our healthy cells [2].  One such alternative that has been gaining traction is the Rosary Pea (Abrus precatorius), a flowering plant with bright red seeds and a toxic dose of 5mg/kg in mice. It is seventy times more deadly than ricin, one of the most toxic substances to ever exist [3]. Despite the toxicity, the chemicals isolated from the leaves, stem, and root of Abrus precatorius have anti-tumor, anti-microbial, antioxidant, and anti-inflammatory properties [4] which make it a tempting drug in the fight against cancer. 

One such chemical is Abrus Agglutinin (AGG): a type II ribosome-inactivating protein that primarily kills cells through mitochondrial-dependent apoptosis—or death by disrupting the mitochondria. For the last fifteen years, a group of researchers in India have been studying the effects of AGG on numerous different cancer types and measuring its impact on tumor cells at a molecular level. They have mainly focused on observing changing protein levels in tumor cells that indicate apoptosis and measuring extracellular cytokine levels to see how AGG stimulates the immune system. Additionally, they studied the in vivo effects of AGG in mice to test the effectiveness and toxicity of the drug against tumor and normal cells under realistic conditions. Therefore, this review analyzes the intracellular changes, extracellular changes, and selective cytotoxicity of AGG on cancer cells, focusing on their studies that included both in vitro and in vivo experiments to gain a fuller understanding of the effect of AGG on cancer cells and its potential as a chemotherapeutic agent. 

Internal Cell Signaling Changes

AGG induces apoptosis in tumor cells through multiple cell signaling avenues, allowing it to downregulate cell cycle proliferation proteins and stop tumor progression through multiple pathways [5]. This section will review the most common proteins affected and their impact on tumor cells starting with the Bax/Bcl-2 proteins.

Many of the studies on AGG have cited these two proteins as major players in apoptosis [6,7,8,9,10]. Bcl-2 is an anti-apoptotic—or pro-life—protein while Bax is a pro-apoptotic—or pro-death—protein that binds the mitochondria in response to stress to induce apoptosis [11]. To avoid potentially dangerous mutations from stressors, the cell releases Bax and induces apoptosis. Because of their importance in apoptosis, the researchers measured the Bax/Bcl-2 ratio before and after the addition of AGG to the tumor cell lines in vitro to see if AGG induced cell death. These studies showed that the proliferating Bcl-2 levels decreased and apoptotic Bax levels increased in MDA-MB-231 breast cancer [6], Dalton’s lymphoma [7], FaDu squamous cell carcinoma [9,10], and HepG2 hepatocellular carcinoma cell lines [8]. Increased Bax levels in cancer cells demonstrate that the addition of AGG induces apoptosis in these cells.  Additionally, decreased Bcl-2 levels indicate AGG’s role in reducing cancer cell viability. 

Secondly, AGG activates the caspases present in tumor cells [5,6,7,8,9,10]. Caspases are a group of inactive proteases–or protein destroyers–that become activated during apoptotic conditions, like increased Bax levels [8], and help the cell continue cell death [12]. The studies measured caspase levels before and after the addition of AGG. They found increases in caspase 3 [5,6,7,8,9,10], caspase 7 [8], caspase 8 [6,8], and caspase 9 [6,8]. These in-vitro studies also showed that the caspases increased in a dose-dependent manner with AGG indicating that the caspases directly contribute to apoptosis. In addition to increased Bax levels, these caspases can also become activated when AGG inactivates molecules that help cells survive and replicate—like Hsp90 and Akt protein kinase [5,6,8]. When AGG decreases Hsp90 and Akt, it alerts caspases that a stressful event is occurring and activates them [8]. Activated caspases will cut proteins involved in keeping cells alive, effectively killing the cells. Therefore, because AGG can activate caspases through multiple pathways to initiate cell death, this makes it a promising drug to treat cancer. 

There is also evidence that AGG directly interferes with the cell cycle by halting the cell in the cell resting phase (G0/G1) or genome duplication phase (S) [7,13]. It does this by decreasing the concentration of proteins that control the cell cycle and creating a situation called mitotic catastrophe where the cells fail to divide correctly and die [9]. A study on the effect of AGG on Dalton’s Lymphoma in mice measured the number of tumor cells in each phase of the cell cycle before and after administering AGG. SubG1 cells—a unique phase that means the cell cycle has failed to begin another cell cycle—increased by 13.42%, 48.9%, and 93.2% at doses of 100, 200, and 500 μg/kg of AGG [7]. These doses are significantly less than the toxic dose of 5mg/kg, demonstrating that AGG could be an incredibly beneficial cancer drug. Another study observed the same increase in SubG1 cells but also went a step further by looking at the change in Cyclin E and Cyclin B1 in FaDu carcinoma cells. Cyclin E and B1 are important proteins that help cells transition to the next stages of the cell cycle so they can give information about the state of cell replication. The study found that their levels decreased after being treated with AGG [9] which means that the cells were unable to move through all the stages of the cell cycle. Increased SubG1 cells and decreased cyclins show that AGG induces apoptosis by affecting the proteins cells need to divide, destabilizing the cells’ structure, and leaving them in a suppressed or unproductive state. 

All of these independent and interlocking mechanisms to initiate cell death are important because having many pathways to the same goal helps AGG avoid any mutated roadblocks such as nonfunctional Bax or caspase 3 proteins [5]. When a target molecule or receptor is mutated, drugs cannot exploit their functions as easily and the tumor progresses, as seen in triple-negative breast cancer [14]. Having multiple avenues to target tumor cells such as Bax/Bcl-2, caspases, and cell cycle inhibition makes AGG a beneficial molecule in the fight against cancer in-vitro, and more studies are needed to further confirm the possible application in humans. 

External Cell Signaling Changes

Internal cell signaling changes are not the only way AGG can induce apoptosis, however. It can also target the host’s immune system to help it fight tumor cells [6,7,10,13,15,16] as well as increase the concentration of reactive oxygen species (ROS) present around the cell [5,6,9,10]. This section will focus on the effect AGG has on the immune system and ROS concentration by looking at its apoptotic effect on tumor cells. 

Many modern cancer treatments can activate the immune system to target cancer cells. In a recent study, researchers found that the addition of AGG to mice generated a humoral and cellular immune response in mice with and without cancer, increasing the activity of T cells [16], macrophages, and natural killer (NK) cells [6,13]. Even when AGG was given to mice for only a week, these cell concentrations increased [13]. Another way the studies checked the immune system's activity was by measuring cytokine levels [13,15,16]. Cytokines are signaling molecules that interact with immune cells to help them grow, divide, and react. After AGG, there were noticeable increases in the cytokines IL-2 and INFγ [13,15,16,]. These are cytokines associated with T cells that help B cells create antibodies and divide [17]. Immune system activation by AGG increases the chances that an immune cell will recognize a tumor cell as unnatural and destroy it. This makes AGG a useful chemical in cancer treatment.

However, there was some disagreement with the cytokine TNFɑ, an inflammatory signaling molecule that promotes apoptosis, which increased in vitro [15] but did not change in vivo [16]. This could be because animal models can react differently than cells in sterile laboratory conditions so this discrepancy is something to keep in mind when moving forward with experiments. Additionally, it’s worth mentioning that AGG was unable to increase immune responses out of thin air. In one study, it needed a pre-existing supply of macrophages—a type of immune cell that eats dead or foreign cells. With mice deficient in macrophages, AGG did not help create more of them [16]. If a specific type of cancer downregulated the immune system to survive, this means AGG would have a hard time targeting cancerous cells with macrophages and the cancer might proliferate. It is also important to note that immune responses can have both a negative and positive effect on the body because our healthy cells can be accidentally attacked along with tumor cells. However,  no negative effect was seen in the study [16]. Many chemotherapeutic drugs have side effects and consequences. Activating the immune system for long periods can be harmful to patients but because treatment with AGG would be short-term and mice suffered no side effects, the benefits outweigh the risk. Additionally, because many cancers downregulate the immune system [13], using a drug like AGG that can augment it is useful. This can increase the odds a tumor cell is recognized and removed. 

Along with increasing the immune response, AGG also increases the concentration of reactive oxygen species (ROS) [5,6,9,10] which are toxic molecules that exist naturally in our body but can cause death in high, unregulated concentrations. One of the ways ROS damages cells is through DNA breaks that lead to p73-mediated apoptosis [9]. This is useful because while many chemotherapeutic drugs for head cancers target p53,  it is a commonly mutated protein. AGG, on the other hand, uses ROS to target p73—a rarely mutated protein in the same family as p53—and allows AGG to kill through this alternative pathway [9]. They also discovered that ROS accumulation activates ATM; a protein that stops the cell cycle when the damage starts to occur and will lead to apoptosis as tested in FaDu carcinoma cells [9]. Additionally, ROS accumulation is linked to caspase 8 activation [6] which leads to cell death. Overall, because ROS can induce apoptosis through a reliable protein and activate apoptotic proteins like ATM and caspase 8, AGG-induced ROS generation is a convenient drug in cancer treatment. 

There are many ways cells can die and AGG exploits multiple different pathways simultaneously. By activating and augmenting the cells in our immune system and increasing toxic oxygen species at the same time, AGG can effectively begin apoptosis in cancerous cells. Even though there are some risks associated with the immune system and ROS activation, successful in vivo mouse studies and limited exposure to these effects make AGG a good chemotherapeutic candidate. 

Selective Cytotoxicity

When choosing a chemotherapeutic drug, it is vitally important that the drug doesn’t harm the host’s healthy cells and is well below the toxic dose. This section first outlines the effect of AGG on normal cells in mice and then on normal cells in vitro. 

An important way researchers test the safety of a drug is through animal experiments. For AGG, the toxic dose is 5mg/kg meaning for every kilogram of body weight, 5mg of AGG needs to be ingested to kill the organism. For every experiment, the therapeutic dose was at or below 2mg/kg [5,6,7,8,9,10,13,16,15] in vivo meaning that the benefits of AGG could be seen without the toxic and deadly side effects. That being said, only a few studies monitored the health of the mouse and they mostly used weight and median lifespan to measure it. The rest of the studies either didn’t mention it or only tested AGG against normal cells in vitro. Despite this lack of data, studies that did include mouse health noticed no negative side effects of taking AGG at doses below 2mg/kg [7,8,13]. There was no unusual weight loss or gain in one study [8] and others measured an increase in median lifespan of 4 days [13] and 20 days [7] when taking AGG compared to mice without treatment. However, one study found that a dose of 2mg/kg started to induce negative effects–like tissue damage–in mice [7] which could be a problem if this drug wants to progress to human trials. One possible reason for this discrepancy is that the studies used different cancer cell lines in their experiments which could have varying resistance to AGG. 

The other way researchers can test the selective cytotoxicity of a drug is in vitro by exposing normal versions of cancer cells to AGG and measuring the apoptotic effects [5,6,7,8]. Studies found that normal cells remained largely unaffected by AGG and, overall, AGG had less specificity towards them [5,6,8]. Unfortunately, no cancer treatment is without risks or side effects. Even when selective towards cancerous cells, increasing ROS and immune activity is dangerous. Additionally, mouse models don’t last long. Half of the studies lasted less than a month [6,8,9], with the other half lasting only a week [5,7,13,15,16] before the animal was sacrificed. This makes it difficult to determine if AGG could have negative effects in the future or with long-term use like how it would be used in humans. However, AGG remains a strong chemotherapeutic candidate due to its selectivity and limited negative effects in mice. 

From the studies, AGG seems to be effective at safe doses but might not be useful for every type of cancer because it could require higher, unsafe doses in specific situations. The mice used in the study showed almost no negative side effects such as weight loss nor did they die earlier than mice without treatment. Longer mouse studies are needed to confirm the future implications of using AGG but so far, the results look promising. 

Conclusion

Abrus Agglutinin shows potential as a chemotherapeutic agent in treating cancer through intracellular changes, extracellular changes, and selective cytotoxicity. AGG targets cancerous cells and induces them to initiate apoptosis through increasing pro-apoptotic Bax proteins, caspases, the immune system, and ROS while down-regulating pro-survival Akt, cyclins, and Bcl-2 proteins. It shows strong selective cytotoxicity against many cancer cell lines below the toxic dose and almost no toxic effects in mice. However, because mouse models are short-lived and there are dangers to inducing ROS and immune effects, longer in vivo studies are needed to solidify AGG as a beneficial chemotherapeutic agent for human use.

About the Author: Mia Karlsson

Mia Karlsson is a fourth-year undergraduate student majoring in Molecular and Medical Microbiology. She chose microbiology to dive deep into the details of disease, viruses, and the immune system and explore how everything we can’t see works. She chose to write this paper because of the fascinating dichotomy between cancer treatment and poisonous plants and wanted to learn more about it. In her spare time, she makes surreal, eccentric art and helps run an art fundraising club on campus called Cherry Tea Collective that raises money for a different charity each year. She hopes people come away from reading the paper with a broader understanding of cancer, research, and the unique capabilities of poisonous plants.

Author’s Note

There has always been something about poisonous plants that fascinated me. How something that appears so harmless and even beautiful can disrupt our cells at such small doses. This paper originally started as the history of the toxic plant Belladonna and the effect that it has on our cells; a deep dive into its poison. But as I tried to gather papers about Belladonna, I noticed that a lot of the research on poisonous plants wasn’t on how they killed but on how they saved. Not just Belladonna, but most poisonous plants had pockets of research dedicated to their health benefits and uses in society; it was fascinating to learn about. When I settled on the Rosary Pea as my toxic plant of interest, I loved picking apart the details and uncovering the puzzle of how exactly the toxin works. The main idea is deceptively simple: if the toxin hurts our cells and cancer cells are similar to our cells, then the Rosary Pea can be exploited against cancer cells. When people read this paper, I hope they see just how special, complicated, and valuable poisonous plants can be and feel a deeper appreciation for plants.

References

1. Siegel RL, Miller KD, Fuchs HE, Jemal A. 2022. Cancer statistics. CA: A Cancer Journal for Clinicians. 72(1):7–33. 

2. Zhu L, Lin M. The Synthesis of Nano-Doxorubicin and its Anticancer Effect. Anti-Cancer Agents in Medicinal Chemistry. 2021;21(18):2466–2477.

3. Peng J, Wu J, Shi N, Xu H, Luo L, Wang J, Li X, Hu X, Feng J, Li X, and others. 2022. A Novel Humanized Anti-Abrin A Chain Antibody Inhibits Abrin Toxicity In Vitro and In Vivo. Frontiers in Immunology. Volume 13. doi: 10.3389%2Ffimmu.2022.831536

4. Qian H, Wang L, Li Y, Wang B, Li C, Fang L, Tang L. 2022. The traditional uses, phytochemistry and pharmacology of Abrus precatorius L.: A comprehensive review. Journal of Ethnopharmacology. 296:115463. 

5. Behera B, Mishra D, Roy B, Devi KSP, Narayan R, Das J, Ghosh SK, Maiti TK. 2014. Abrus precatorius agglutinin-derived peptides induce ROS-dependent mitochondrial apoptosis through JNK and Akt/P38/P53 pathways in HeLa cells. Chemico-Biological Interactions. 222:97–105. 

6. Bhutia SK, Behera B, Das DN, Mukhopadhyay S, Sinha N, Panda PK, Naik PP, Patra SK, Mandal M, Sarkar S, and others. 2016. Abrus agglutinin is a potent anti-proliferative and anti-angiogenic agent in human breast cancer. International Journal of Cancer. 139(2):457–466. 

7. Bhutia SK, Mallick SK, Maiti S, Maiti TK. 2008. Antitumor and proapoptotic effect of Abrus agglutinin derived peptide in Dalton’s lymphoma tumor model. Chemico-Biological Interactions. 174(1):11–18. 

8. Mukhopadhyay S, Panda PK, Das DN, Sinha N, Behera B, Maiti TK, Bhutia SK. 2014. Abrus agglutinin suppresses human hepatocellular carcinoma in vitro and in vivo by inducing caspase-mediated cell death. Acta Pharmacologica Sinica. 35(6):814–824. 

9. Sinha N, Panda PK, Naik PP, Das DN, Mukhopadhyay S, Maiti TK, Shanmugam MK, Chinnathambi A, Zayed ME, Alharbi SA, and others. 2017. Abrus agglutinin promotes irreparable DNA damage by triggering ROS generation followed by ATM-p73 mediated apoptosis in oral squamous cell carcinoma. Molecular Carcinogenesis. 56(11):2400–2413. 

10. Sinha N, Panda PK, Naik PP, Maiti TK, Bhutia SK. 2017. Abrus agglutinin targets cancer stem-like cells by eliminating self-renewal capacity accompanied with apoptosis in oral squamous cell carcinoma. Sage J [internet]. 39(5). doi:10.1177/1010428317701634 

11. Hardwick JM, Soane L. 2013. Multiple Functions of BCL-2 Family Proteins. Cold Spring Harbor Perspectives in Biology [internet]. 2013;5(2). doi: 10.1101/cshperspect.a008722 

12. Shi Y. 2004. Caspase activation, inhibition, and reactivation: A mechanistic view. Protein Science : A Publication of the Protein Society. 13(8):1979–1987. 

13. Ghosh D, Maiti T. 2007. Immunomodulatory and anti-tumor activities of native and heat denatured Abrus agglutinin. Immunobiology. 212(7):589–599. 

14. Triple-Negative Breast Cancer: Why Is It Harder to Treat? Willamette Valley Cancer Institute and Research Center. Accessed November 15, 2023. Available from: https://www.oregoncancer.com/blog/why-is-triple-negative-breast-cancer-harder-t o-treat 

15. Behera B, Devi KSP, Mishra D, Maiti S, Maiti TK. 2014. Biochemical analysis and antitumour effect of Abrus precatorius agglutinin derived peptides in Ehrlich’s ascites and B16 melanoma mice tumour model. Environmental Toxicology and Pharmacology. 38(1):288–296. 

16. Ghosh D, Maiti T. 2007. Effects of native and heat-denatured Abrus agglutinin on tumor-associated macrophages in Dalton’s lymphoma mice. Immunobiology. 212(8):667–673. 

17. Smith KM, Pottage L, Thomas ER, Leishman AJ, Doig TN, Xu D, Liew FY, Garside P. 2000. Th1 and Th2 CD4+ T cells provide help for B cell clonal expansion and antibody synthesis in a similar manner in vivo. Journal of Immunology.165(6):3136–3144.