Receptor tyrosine kinase-like orphan receptor 1: A novel antitumor target in gastrointestinal cancers.

Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is a member of the type I receptor tyrosine kinase family. ROR1 is pivotal in embryonic development and cancer, and serves as a biomarker and therapeutic target. It has soluble and membrane-bound subtypes, with the latter highly expressed in tumors. ROR1 is conserved throughout evolution and may play a role in the development of gastrointestinal cancer through multiple signaling pathways and molecular mechanisms. Studies suggest that overexpression of ROR1 may increase tumor invasiveness and metastasis. Additionally, ROR1 may regulate the cell cycle, stem cell characteristics, and interact with other signaling pathways to affect cancer progression. This review explores the structure, expression and role of ROR1 in the development of gastrointestinal cancers. It discusses current antitumor strategies, outlining challenges and prospects for treatment.

Multifaceted p21 in carcinogenesis, stemness of tumor and tumor therapy.

Cancer cells possess metabolic properties that are different from those of benign cells. p21, encoded by CDKN1A gene, also named p21Cip1/WAF1, was first identified as a cyclin-dependent kinase regulator that suppresses cell cycle G1/S phase and retinoblastoma protein phosphorylation. CDKN1A (p21) acts as the downstream target gene of TP53 (p53), and its expression is induced by wild-type p53 and it is not associated with mutant p53. p21 has been characterized as a vital regulator that involves multiple cell functions, including G1/S cell cycle progression, cell growth, DNA damage, and cell stemness. In 1994, p21 was found as a tumor suppressor in brain, lung and colon cancer by targeting p53 and was associated with tumorigenesis and metastasis. Notably, p21 plays a significant role in tumor development through p53-dependent and p53-independent pathways. In addition, expression of p21 is closely related to the resting state or terminal differentiation of cells. p21 is also associated with cancer stem cells and acts as a biomarker for such cells. In cancer therapy, given the importance of p21 in regulating the G1/S and G2 check points, it is not surprising that p21 is implicated in response to many cancer treatments and p21 promotes the effect of oncolytic virotherapy.

Complete eradication of xenograft hepatoma by oncolytic adenovirus ZD55 harboring TRAIL-IETD-Smac gene with broad antitumor effect.

Cancer-targeting dual-gene virotherapy (CTGVT-DG) is an important modification of CTGVT, in which two suitable genes are used to obtain an excellent antitumor effect. A key problem is to join the two genes to form one fused gene, and then to clone it into the oncolytic viral vector so that only one investigational new drug application, instead of two, is required for clinical use. Many linkers (e.g., internal ribosome entry site) are used to join two genes together, but they are not all equally efficacious. Here, we describe finding the best linker, that is, sequence encoding the four amino acids IETD, to join the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene and the second mitochondria-derived activator of caspase (Smac) gene to form TRAIL-IETD-Smac and inserting it into oncolytic viral vector ZD55 to construct ZD55-TRAIL-IETD-Smac, which matched ZD55-TRAIL plus ZD55-Smac in completely eliminating xenograft hepatoma. ZD55-TRAIL-IETD-Smac works by quantitative cleavage at IETD↓by inducing caspase-8; activation or inhibition of caspase-8 could up- or downregulate cleavage, respectively. The cleaved product, TRAIL-IETD, does not affect the function of TRAIL. Numerous experiments have shown that the combined use of ZD55-TRAIL plus ZD55-X could completely eradicate many xenograft tumors, and therefore the IETD is potentially a useful linker to construct many antitumor drugs, for example, ZD55-TRAIL-IETD-X, where X has a compensative or synergetic effect on TRAIL. We found that the antitumor effect of ZD55-IL-24-IETD-TRAIL also has an equivalent antitumor effect compared with the combined use of ZD55-IL-24 plus ZD55-TRAIL, because ZD55-IL-24 could also induce caspase-8. This means that IETD, as a two-gene linker, may have broad use.

The antitumor efficacy of IL-24 mediated by E1A and E1B triple regulated oncolytic adenovirus.

BACKGROUND: IL-24 (interleukin-24) is a promising, multi-functional anti-cancer agent able to selectively induce tumor cell apoptosis while sparing normal cells. Additionally, IL-24 can enhance the immune response to tumors and suppress tumor angiogenesis. In this study, we introduced IL-24 into the oncolytic adenovirus, Ad·sp·E1A((Δ24))·E1B((Δ55))·IL-24. in which E1A was engineered to target Rb (retinoblastoma) deficient or dysfunctional tumors. The survivin promoter (sp), was used to drive expression of IL-24, thereby allowing it to target most tumors. Finally, the 55 KDa gene of E1B was also deleted, thereby preventing replication in normal cells. RESULTS: Ad·sp·E1A((Δ24))·E1B((Δ55))·IL-24 showed enhanced antitumor effects over the E1, singly regulated oncolytic adenovirus, ONYX-015, in in vitro experiments. Furthermore, Ad·sp·E1A((Δ24))·E1B((Δ55))·IL-24 could effectively inhibit the progression of NCI-H460 lung carcinoma xenografts in nude mice. METHODS: The antitumor effect of Ad·sp·E1A((Δ24))·E1B((Δ55))·IL-24 was assessed by MTT assay and crystal violet staining in a panel of tumor cells. Cell staining and western blotting for caspase activation were used to assess apoptosis. We assessed the antitumor effects of Ad·sp·E1A((Δ24))·E1B((Δ55))·IL-24 in a xenograft model. CONCLUSION: This is the first study to use an E1A and E1B triple regulated oncolytic adenovirus vector carrying IL-24 to treat large tumors. We attained efficient antitumor effects both in vitro and in vivo, which provides an experimental foundation for clinical cancer therapy.

Comparative proteomic analysis of colon cancer cells in response to oxaliplatin treatment.

Colon cancer is one of the most common malignancies in the world. Oxaliplatin, a third-generation platinum compound, is widely used in clinical chemotherapy of colon cancer. Although the mechanisms of the antitumor effect of Oxaliplatin have been investigated in recent years, the proteomic changes that are associated with the cellular response to this compound are poorly understood. In this study, we performed a comparative proteomic analysis to survey the global changes in protein expression levels after Oxaliplatin treatment in three colon cancer cell lines: HT29, SW620, and LoVo. Two-dimensional gel electrophoresis coupled with MALDI-TOF/TOF mass spectrometry revealed 57, 48, and 53 differentially expressed proteins in the three cell lines (HT29, SW620 and LoVo, respectively) after Oxaliplatin treatment. Of these proteins, 21 overlapped among all three cell lines. These overlapping proteins participate in many cellular processes, such as apoptosis, signal transduction, transcription and translation, cell structural organization, and metabolism. Additionally, the expression levels of ezrin (EZRI), heat-shock protein beta-1 (HSPB1), translationally controlled tumor protein (TCTP), and cell division control protein 2 homolog (CDC2) were confirmed by immunoblotting. This is the first direct proteomic analysis of Oxaliplatin-treated colon cancer cells. Several interesting proteins that we found warrant further investigation owing to their potential significant functions in the antitumor effect of Oxaliplatin.

Mutational analysis of the absolutely conserved B8Gly: consequence on foldability and activity of insulin.

B8Gly is absolutely conserved in insulins during evolution. Moreover, its corresponding position is always occupied by a Gly residue in other members of insulin superfamily. Previous work showed that Ala replacement of B8Gly significantly decreased both the activity and the foldability of insulin. However, the effects of substitution are complicated, and different replacements sometimes cause significantly different results. To analyze the effects of B8 replacement by different amino acids, three new insulin/single-chain insulin mutants with B8Gly replaced by Ser, Thr or Leu were prepared by protein engineering, and both their foldability and activity were analyzed. In general, replacement of B8Gly by other amino acids causes significant detriment to the foldability of single-chain insulin: the conformations of the three B8 mutants are essentially different from that of wild-type molecules as revealed by circular dichroism; their disulfide stabilities in redox buffer are significantly decreased; their in vitro refolding efficiencies are decreased approximately two folds; the structural stabilities of the mutants with Ser or Thr substitution are decreased significantly, while Leu substitution has little effect as measured by equilibrium guanidine denaturation. As far as biological activity is concerned, Ser replacement of B8Gly has only a moderate effect: its insulin receptor-binding activity is 23% of native insulin. But Thr or Leu replacement produces significant detriment: the receptor-binding potencies of the two mutants are less than 0.2% of native insulin. The present results suggest that Gly is likely the only applicable natural amino acid for the B8 position of insulin where both foldability and activity are concerned.

Replacement of the interchain disulfide bridge-forming amino acids A7 and B7 by glutamate impairs the structure and activity of insulin.

Insulin contains three disulfide bonds, one intrachain bond, A6-A11, and two interchain bonds, A7-B7 and A20-B19. Site-directed mutagenesis results (the two cysteine residues of disulfide A7-B7 were replaced by serine) showed that disulfide A7-B7 is crucial to both the structure and activity of insulin. However, chemical modification results showed that the insulin analogs still retained relatively high biological activity when A7Cys and B7Cys were modified by chemical groups with a negative charge. Did the negative charge of the modification groups restore the loss of activity and/or the disturbance of structure of these insulin analogs caused by deletion of disulfide A7-B7? To answer this question, an insulin analog with both A7Cys and B7Cys replaced by Glu, which has a long side-chain and a negative charge, was prepared by protein engineering, and its structure and activity were analyzed. Both the structure and activity of the present analog are very similar to that of the mutant with disulfide A7-B7 replaced by Ser, but significantly different from that of wild-type insulin. The present results suggest that removal of disulfide A7-B7 will result in serious loss of biological activity and the native conformation of insulin, even if the disulfide is replaced by residues with a negative charge.

Peptide models of four possible insulin folding intermediates with two disulfides.

The single-chain insulin (PIP) can spontaneously fold into native structure through preferred kinetic intermediates. During refolding, pairing of the first disulfide A20-B19 is highly specific, whereas pairing of the second disulfide is likely random because two two-disulfide intermediates have been trapped. To get more details of pairing property of the second disulfide, four model peptides of possible folding intermediates with two disulfides were prepared by protein engineering, and their properties were analyzed. The four model peptides were named [A20-B19, A7-B7]PIP, [A20-B19, A6-B7]PIP, [A20-B19, A6-A11]PIP, and [A20-B19, A7-A11]PIP according to their remaining disulfides. The four model peptides all adopt partially folded structure with moderate conformational differences. In redox buffer, the disulfides of the model peptides are more easily reduced than those of the wild-type PIP. During in vitro refolding, the reduced model peptides share similar relative folding rates but different folding yields: The refolding efficiency of the reduced [A20-B19, A7-A11]PIP is about threefold lower than that of the other three peptides. The present results indicate that the folding intermediates corresponding to the present model peptides all adopt partially folded conformation, and can be formed during PIP refolding, but the chance of forming the intermediate with disulfide [A20-B19, A7-A11] is much lower than that of forming the other three intermediates.