INTRODUCTION:

Ribosome-inactivating proteins (RIPs) are toxins that are apt to permanently inhibit the translation or the synthesis of protein. These proteins are widely distributed in nature, particularly in plants. RIPs in plants are categorized into three types: (i) Type I, composed of a single polypeptide chain (monomeric protein) of approximately 30 kDa, consists of an N-terminal, an active domain, and a C-terminal (1); (ii) Type II is a heterodimeric protein consisting of an A chain (an N-terminal and an active domain, functionally equivalent to the type I polypeptide), linked to a lectin-like B chain by a disulfide bridge, and a C-terminal¹.

The lectin binds to glycoproteins or glycolipids to promote a toxin-endocytosis that leads to its higher toxicity compared to the type I^2,3(iii) Type III consists of an N-terminal domain closely related to the A chain of RIPs and linked to an unrelated C-terminal domain with unknown function⁴.In cell walls or membranes, lectin binds to sugar moieties and thus alters the physiology of the membrane to induce cell agglutination, mitosis, or other biochemical changes⁵.

RIPs are officially defined as rRNA N-glycosidases (EC 3.2.2.22). These proteins catalytically inactivate eukaryotic and prokaryotic ribosomes, and selectively break the bond between adenine and ribose from the universally conserved α-sarcin/ricin loop (SRL) (Figure 1) of the large rRNA^4,2. The SRL is a highly conserved sequence found in the RNA of all large ribosomal subunits. The structural stability of the SRL is constructed mostly by π-π interactions. Removal of the key adenine at the terminal of the loop by cytotoxins can destabilize this interaction⁶. The SRL is an assembly of two secondary structure motifs: an internal loop (bulged G) and a hairpin loop (GNRA tetraloop)⁷.

The α-sarcin/ricin loop or SRL is critical to initiate GTP hydrolysis on the elongation factor Tu (EF-Tu) and the elongation factor G (EF-G), which delivers aminoacyl-transfer RNAs (tRNAs) to the ribosome. As the SRL is involved in the elongation of the factor bindings, the modified ribosomes are catalytically inactive and unable to support protein synthesis^8,9. Moreover, a study on the activated conformation of EF-Tu (PDB ID: 4V5L https://www.rcsb.org/structure/4V5L) revealed that the activation is due to a critical and conserved interaction of the amino acid His with SRL A2662 of the 23S ribosomal RNA⁹. Histidine residue (His84 in EF-Tu, His87 in EF-G, or His61 in SelB, or His108 in eEF2) plays an important role in catalysis. This histidine may occur in two conditions: (i) a flipped-out conformation (a distance away from the γ-phosphate of GTP) which is inactive; and (ii) an active flipped-in conformation (approaching to the γ-phosphate of GTP)⁶.

The SRL is a highly conserved sequence found in the RNA of all large ribosomal subunits. The cytotoxins SRL both inactivate ribosomes by cleaving a single bond in that loop and stop the translation¹⁰.

Well-known examples of RIPs contained in plants are ricin, abrin, and saporins¹¹. This article is devoted to reviewing the anticancer activity of ricin (Ricinus communis L., Euphorbiaceae) and the role of various cytotoxicity enhancers.

RICIN:

The dark brown seeds of Ricinus communis L. or castor bean (Euphorbiaceae) (Figure 2) contain 54% of oil which consists of esters of palmitic (1.2%), stearic (0.7%), arachidic (0.3%), hexadecanoic (0.2%), oleic (3.2%), linoleic (3.4%), linolenic (0.2%), ricinoleic (89.4%), and dihydroxy stearic acid¹². Ricin isolated from Ricinus communis L. seeds (Figure 2) is a heterodimeric two-domain polypeptide protein that includes an enzymatically active domain (chain A) of approximately 30 kDa and a slightly bigger lectin-like B domain of about 35 kDa. The B-lectin domain possesses sugar-binding properties³. The chain A (length 267) is glycosidase that removes a specific adenine residue from an exposed loop of the 28S rRNA at the SRL, which leads to cleavage of rRNA (http://www.rcsb.org/pdb/protein/P02879). The cytotoxins α-sarcin and ricin both work to inactivate the ribosomes by breaking a single bond in that loop. Once the SRL is invaded, the ribosomes can not interact with elongation factors properly, and the translation terminates¹⁰. The inhibition of protein synthesis in the eukaryotic cells by RIPs is accomplished by enzymatically altering the 28S rRNA of the large 60S ribosomal subunit¹¹.

Figure 2. (a) Ricinus communis seeds; (b) The 3D structure of plant cytotoxin ricin (PDB ID: 2AAI downloades from http://www.rscb.org//3d-view/2AAI/1) ¹³. The A chain acts as glycosidase that removes a specific adenine residue from an exposed loop of 28r RNA leading to rRNA breakge.

2,3 α-sarcin breaks the phosphodiester bond between G2661 and A2662, while ricin hydrolytically cleaves the β-N-glycosidic bond of A2660 in the SRL^8,6. The A chain can inactivate thousands of ribosomes/minute, hence a single A chain can destroy an animal cell. The B-lectin chain (length 262) binds to beta-D-galactopyranoside moieties on cell surface glycoproteins and glycolipids and facilitates the entry into the cell of the A chain; B-lectin chain is also responsible for cell agglutination (http://www.rcsb.org/pdb/protein/P02879). When A chain is separated from the B-lectin chain, the arginine residues located at the dimeric interface domain is exposed to the solvent and serves as an interaction platform for P1/P2 protein dimers (P stalk proteins). The A chain-stalk interaction stimulates the toxin to initiate its enzymatic activity by pointing the active site of A chain (opposite to the arginine interface) toward the SRL¹⁴.

CROSSTALK BETWEEN CANCER, APOPTOSIS, AND AUTOPHAGY:

Cancer is a major illness in the world, affecting many people around the world, as many individuals are affected by breast cancer , lung cancer, cervical cancer, bone cancer, etc^15,16. Cancer can be identified as malignant tissue growth or enlargement due to unlimited and uncontrolled mitotic divisions of certain cells that invade surrounding tissues ¹⁷. Due to the effects of carcinogenes, such as cigarette smoke, radiation, chemicals, or infectious agents, these anomalies can occur^18,19. Other cancer-promoting genetic defects can occur spontaneously or are inherited by errors in DNA replication, and are thus present from birth in all cells¹⁸. Genes that control cell growth and differentiation must be altered in order for a normal cell to turn into a cancer cell. At several levels, genetic modifications may occur, from the benefit or loss of whole chromosomes to a mutation affecting a single nucleotide of DNA²⁰.

Depending on the strength and furation of the wound, the ATP depletion rates and the availability of caspase, the process and morphology of apoptosis may occur simultaneously. Apoptosis can be caused by various injuries such as pressure, hypoxia, radiation, and low-dose anticancer drugs²¹. Cell death caused by anticancer drugs is closely associated with an increase in apoptosis via the caspase-dependent pathway¹⁷. The apoptotic process is of widespread biological significance, involving, for example, the growth, differentiation, proliferation/homeostatis, immune system control and function, and the removal of defects and thus harmful cells^21,22. Caspase 3 was reported as negatively corelated with apopptosis in retinonlastoma (malignant retinal tumor)²³ .

In a number of pathological conditions, malfunction or dysregulation of the apoptotic program is thus suggested. Cancer, autoimmune diseases and the spread of viral infections may result in apoptosis defects, whereas neurogenerative disorders, AIDS and ischaemic diseases are caused by excessive apoptosis¹⁷.

The underlying mechanism of the cell that hits the unwanted components is autophagy. When detaching unique organelles, it can be selective or non-selective. Homeostatis is protected by protein degradation. Autophagy helps cells to insist on stress such as nutrient withdrawal from an external environment and also helps them to avoid internal stress such as an accumulation of destructive organelles²⁴.

THE ANTICANCER ACTIVITY OF RICIN AND THE CYTOTOXICITY ENHANCERS:

The capability of inhibiting protein synthesis by a single molecule of ricin in the cytosol has brought a wide exploration of this plant toxin for its anticancer activity. Ricin was described could strongly induce the occurrence of apoptosis in HeLa cells (IC₅₀ for cell viability = 1μg/mL)²⁵. Ricin could induce caspase activation and also activated c-Jun N-terminal kinase (JNK) and p38 signaling pathways in the immortalized, nontransformed epithelial cell lines (MAC-T)²⁶.

Nonetheless, the utility of ricin as an anticancer agent is limited due to the non-selectivity of the B-lectin chain. Liposome has been used as a delivering-vehicle, but this vehicle significantly reduced the cytotoxicity of ricin²⁷.

Interestingly, a previous study reported that ricin-liposome complexes could penetrate the SKMEL-28 melanoma cells via endocytosis. The ricin-liposome complexes demonstrated high cytotoxicity (IC₅₀ ranged from 62.4ng/mL to 604.3ng/mL) towards SKMEL-28 cells at 36 hours post-treatment²⁸. Another study revealed that liposomal monensin could be employed to enhance the cytotoxicity of liposomal ricin in human epidermoid cancer cells^29,30. An entrapment of ricin in folate-tagged-sterically stabilized liposomes in combination with intercalated-monensin may increase the cytotoxicity of liposomal ricin against KB cancer cells³¹.

Moreover, a case study on a 57-year-old male patient with multiple bladder cancer (5 different sizes of tumors) has been reported. This patient has been treated with the conjugation of ricin A-BCMab1 (a particular monoclonal antibody that could recognize the glycosylated-Integrin a3b1 in bladder cancer). After 26 weeks of weekly treatment, the tumors were dissipated ³².

CONCLUSION:

Ricin, isolated from Ricinus communis L. seeds, is a heterodimeric two-domain polypeptide protein that includes an enzymatically active chain A and a lectin-like B domain. A single molecule of this protein is capable to inactivate thousands of ribosomes/minutes and has been proven in inducing apoptosis of cancer cells. The non-selectivity of the B-lectin chain gives rise to the need for a cytotoxicity enhancer, and liposome or its derivatives have been proven as effective.

ACKNOWLEDGEMENTS:

The authors would like to thank the Rector of Universitas Padjadjaran (West Java, Indonesia) for funding the publication fee via the Directorate of Research and Community Engagement. The present work was conducted in the framework of the doctoral dissertation of the first author at the Faculty of Pharmacy, Padjadjaran University, West Java, Indonesia.

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Received on 01.09.2020 Modified on 22.02.2021

Research J. Pharm. and Tech 2022; 15(1):405-408.

DOI: 10.52711/0974-360X.2022.00067