Enhancement of 5-FU sensitivity by the proapoptotic rpL3 gene in p53 null colon cancer cells through combined polymer nanoparticles

Abstract

Colon cancer is one of the leading causes of cancer-related death worldwide and the therapy with 5-fluorouracil (5-FU) is mainly limited due to resistance. Recently, we have demonstrated that nucleolar stress upon 5-FU treatment leads to the activation of ribosome-free rpL3 (L3) as proapoptotic factor. In this study, we analyzed L3 expression profile in colon cancer tissues and demonstrated that L3 mRNA amount decreased with malignant progression and the intensity of its expression was inversely related to tumor grade and Bcl-2/Bax ratio. With the aim to develop a combined therapy of 5-FU plus plasmid encoding L3 (pL3), we firstly assessed the potentiation of the cytotoxic effect of 5-FU on colon cancer cells by L3. Next, 10 μM 5-FU and 2 μg of pL3 were encapsulated in biocompatible nanoparticles (NPs) chemically conjugated with HA to achieve active tumor-targeting ability in CD44 overexpressing cancer cells. We showed the specific intracellular accumulation of NPs in cells and a sustained release for 5-FU and L3. Analysis of cytotoxicity and apoptotic induction potential of combined NPs clearly showed that the 5-FU plus L3 were more effective in inducing apoptosis than 5-FU or L3 alone. Furthermore, we show that the cancer-specific chemosensitizer effect of combined NPs may be dependent on L3 ability to affect 5-FU efflux by controlling P-gp (P-glycoprotein) expression. These results led us to propose a novel combined therapy with the use of 5-FU plus L3 in order to establish individualized therapy by examining L3 profiles in tumors to yield a better clinical outcomes.

Role of uL3 in Multidrug Resistance in p53-Mutated Lung Cancer Cells

“…On the other hand, ABC family proteins, such as Multi Drug Reactivity 1 (MDR1), are related to multiple drug resistance in cancer. We have previously demonstrated that uL3 is able to negatively control Pgp expression in colon cancer cells by regulation of MDR1 mRNA stability [24]. Moreover, we also demonstrated that the loss of uL3 and the consequent upregulation of the cystathionine β synthase (CBS) enzyme correlated with ineffectiveness of 5-FU in lung and colon cancer cell lines [23].…”

“…We have also previously observed a reduction of uL3 levels in Cisplatin resistant A549 cells [25] that, unlike Calu-6 cells, are proficient for p53, suggesting that the loss of uL3 could be considered a general mechanism of multidrug resistance independent of p53 status. Furthermore, we demonstrated that the loss of uL3 was associated with the up-regulation of the MDR1 mRNA level and Pgp transporter encoded by MDR1 in colon cancer cells [24]. According to this, the multidrug resistance in rCalu-6 cells is associated to the high expression of the human MDR1 gene and the Pgp protein levels.…”

“…In conclusion, in this study we provide new insights concerning a rational applicability of the combined treatment based on 5-FU plus uL3 [24] as an individualized therapy for tumors lacking uL3, in an attempt to overcome drug resistance and yield better clinical outcomes.…”

“…We demonstrated that upon ribosomal stress induced by 5-FU, ribosome free uL3 becomes a regulator of p21 [19,20], cystathionine-β-synthase (CBS) [21,22,23], and NFκB [22] expression and mediates apoptosis through the activation of the mitochondrial apoptotic response pathway. Of note, our data indicated that uL3 status is associated to chemoresistance since the loss of uL3 makes chemotherapeutic drugs, such as 5-FU, oxaliplatin (L-OHP), Actinomycin D, and Cisplatin ineffective in colon and lung cancer cells lacking active p53 [21,22,23,24]. According to this, we reported that the resistance of A549 lung cancer cells to Cisplatin correlates to the loss of uL3 expression [25].…”

Oridonin induces Mdm2‐p60 to promote p53‐mediated apoptosis and cell cycle arrest in neuroblastoma

“…Similarly, in TP53 null colon cancer cells, 5‐FU (5‐fluorouracil) can cause nucleolar stress to lead to cell cycle arrest and apoptosis. The nucleolar stress induced by 5‐FU can further activate rpL3 (ribosomal protein L3), which induce the expression of its target gene CDKN1A and regulates it's another target gene CBS (cystathionine‐beta‐synthase) to induce mitochondrial apoptosis characterized by an increase of BAX . This suggests that it is worth to explore whether oridonin can lead to cell cycle arrest and apoptosis via p53‐independent pathways, such as inducing nucleolar stress to regulate ribosomal proteins like rpL3.…”