Effects of Skeleton Structure of Mesoporous Silica Nanoadjuvants on Cancer Immunotherapy.

Mesoporous silica nanoparticles (MSNs) have been widely praised as nanoadjuvants in vaccine/tumor immunotherapy thanks to their excellent biocompatibility, easy-to-modify surface, adjustable particle size, and remarkable immuno-enhancing activity. However, the application of MSNs is still greatly limited by some severe challenges including the unclear and complicated relationships of structure and immune effect. Herein, three commonly used MSNs with different skeletons including MSN with tetrasulfide bonds (TMSN), MSN containing ethoxy framework (EMSN), and pure -Si-O-Si- framework of MSN (MSN) are comprehensively compared to study the impact of chemical construction on immune effect. The results fully demonstrate that the three MSNs have great promise in improving cellular immunity for tumor immunotherapy. Moreover, the TMSN performs better than the other two MSNs in antigen loading, cellular uptake, reactive oxygen species (ROS) generation, lymph node targeting, immune activation, and therapeutic efficiency. The findings provide a new paradigm for revealing the structure-function relationship of mesoporous silica nanoadjuvants, paving the way for their future clinical application.

Heterostructural Nanoadjuvant CuSe/CoSe2 for Potentiating Ferroptosis and Photoimmunotherapy through Intratumoral Blocked Lactate Efflux.

The desirable curative effect in clinical immunotherapy has been challenging due to the immunosuppressive tumor microenvironment (TME) with high lactic acid (LA) metabolism in solid tumors. Although targeting metabolic reprogramming of tumor cells can restore the survival and function of immune cells in the TME, it is also plagued by insufficient immunogenicity. Herein, an activatable immunomodulatory nanoadjuvant CuSe/CoSe2@syrosingopine (CSC@Syro) is constructed for simultaneously relieving immunosuppressive TME and boosting tumor immune response. Specifically, CuSe/CoSe2 (CSC) exhibits TME-activated glutathione (GSH) depletion and hydroxyl radical (•OH) generation for potential ferroptosis. Meanwhile, the remarkable photothermal conversion efficiency and elevated photocatalytic ROS level both promote CSC heterostructures to induce robust immunogenic cell death (ICD). Besides, the loaded syrosingopine inhibitor achieves LA metabolism blockade in cancer cells by downregulating the expression of monocarboxylate transporter 4 (MCT4), which could sensitize ferroptosis by intracellular milieu acidification and neutralize the acidic TME to alleviate immunosuppression. Hence, advanced metabolic modulation confers the potentiated immune infiltration of ICD-stimulated T lymphocytes and further reinforces antitumor therapy. In brief, CSC@Syro-mediated synergistic therapy could elicit potent immunogenicity and suppress tumor proliferation and metastasis effectually by integrating the tumor metabolic regulation and ferroptosis with immunotherapy.

Fe(III)-Naphthazarin Metal-Phenolic Networks for Glutathione-Depleting Enhanced Ferroptosis-Apoptosis Combined Cancer Therapy.

Nowadays, Fenton chemistry-based chemodynamic therapy (CDT) is an emerging approach to killing tumor cells by converting endogenous H2 O2 into cytotoxic hydroxyl radicals (·OH). However, the elimination of ·OH by intracellular overexpressed glutathione (GSH) results in unsatisfactory antitumor efficiency. In addition, the single mode of consuming GSH and undesirable drug loading efficiency cannot guarantee the efficient cancer cells killing effect. Herein, a simple one-step strategy for the construction of Fe3+ -naphthazarin metal-phenolic networks (FNP MPNs) with ultrahigh loading capacity, followed by the modification of NH2 -PEG-NH2 , is developed. The carrier-free FNP MPNs can be triggered by acid and GSH, and rapidly release naphthazarin and Fe3+ , which is further reduced to Fe2+ that exerts Fenton catalytic activity to produce abundant ·OH. Meanwhile, the Michael addition between naphthazarin and GSH can lead to GSH depletion and thus achieve tumor microenvironment (TME)-triggered enhanced CDT, followed by activating ferroptosis and apoptosis. In addition, the reduced Fe2+ as a T1 -weighted contrast agent endows the FNP MPNs with magnetic resonance imaging (MRI) functionality. Overall, this work is the debut of naphthazarin as ligands to fabricate functional MPNs for effectively depleting GSH, disrupting intracellular redox homeostasis, and enhancing CDT effects, which opens new perspectives on multifunctional MPNs for tumor synergistic therapy.

A Robust Oxygen-Carrying Hemoglobin-Based Natural Sonosensitizer for Sonodynamic Cancer Therapy.

The development of novel sonosensitizers with outstanding reactive oxygen (ROS) generation capacity and great biocompatibility poses a significant challenge for the clinical practice of sonodynamic therapy (SDT). In this work, hemoglobin (Hb) with natural metalloporphyrin was first shown to possess great potential as a sonosensitizer. Compared with traditional organic sonosensitizers, Hb had satisfactory sono-sensitizing efficiency because four the porphyrin molecules were separated by four polypeptide chains. This effectively avoided the problem of low ROS quantum yield caused by the stacking of hydrophobic porphyrins. Meanwhile, Hb is an efficient and safe oxygen carrier that may release O2 at hypoxic tumors site, which improved tumor oxygenation and subsequently enhanced SDT efficacy. Therefore, Hb was integrated with zeolitic imidazolate framework 8 (ZIF-8) to form a nanoplatform that demonstrated a potent suppression effect on deep-seated tumors under ultrasound irradiation.

Core-Shell Structured Upconversion/Lead-Free Perovskite Nanoparticles for Anticounterfeiting Applications.

In view of their excellent luminescence properties, nanocrystalline metal halide perovskites have diverse optoelectronic applications, including those related to anticounterfeiting. However, high-quality optical anticounterfeiting typically requires multiple encryptions relying on several optical modes to ensure information security. Herein, an efficient anticounterfeiting strategy based on dual optical encryption is realized by combining up- and downconversion luminescence in a nanocomposite with NaYF4 : Er3+ ,Yb3+ as core and a CsMnCl3 as shell. The emission color of this nanocomposite depends on the penetration depth of incident radiation and can be changed by varying the excitation source (980 nm laser or UV light) to produce different luminescent patterns. This feature allows one to effectively improve the anticounterfeiting index and fabricate professional anticounterfeiting materials.

NIR-Triggered Multi-Mode Antitumor Therapy Based on Bi2 Se3 /Au Heterostructure with Enhanced Efficacy.

Of all the reaction oxygen species (ROS) therapeutic strategies, NIR light-induced photocatalytic therapy (PCT) based on semiconductor nanomaterials has attracted increasing attention. However, the photocatalysts suffer from rapid recombination of electron-hole pairs due to the narrow band gaps, which are greatly restricted in PCT application. Herein, Bi2 Se3 /Au heterostructured photocatalysts are fabricated to solve the problems by introducing Au nanoparticles (NPs) in situ on the surface of the hollow mesoporous structured Bi2 Se3 . Owing to the lower work function of Au NPs, the photo-induced electrons are easier to transfer and assemble on their surfaces, resulting in the increased separation of the electron-hole pairs with efficient ROS generation. Besides, Bi2 Se3 /Au heterostructures also enhance the photothermal efficiency due to the effective orbital overlaps with accelerated electron migrations according to density functional theory calculations. Moreover, the PLGA-PEG and the doxorubicin (DOX) are introduced for photothermal-triggered drug release in the system. The Bi2 Se3 /Au heterostructures also displays excellent infrared thermal (IRT) and computed tomography (CT) dual-modal imaging property for promising cancer diagnosis. Collectively, Bi2 Se3 /Au@PLGA-PEG-DOX exhibits prominent tumor inhibition effect based on synchronous PTT, PCT and chemotherapy triggered by NIR light for efficient antitumor treatment.

Single-Atom Pd Nanozyme for Ferroptosis-Boosted Mild-Temperature Photothermal Therapy.

Photothermal therapy (PTT) is an extremely promising tumor therapeutic modality. However, excessive heat inevitably injures normal tissues near tumors, and the damage to cancer cells caused by mild hyperthermia is easily repaired by stress-induced heat shock proteins (HSPs). Thus, maximizing the PTT efficiency and minimizing the damage to healthy tissues simultaneously by adopting appropriate therapeutic temperatures is imperative. Herein, an innovative strategy is reported: ferroptosis-boosted mild PTT based on a single-atom nanozyme (SAzyme). The Pd SAzyme with atom-economical utilization of catalytic centers exhibits peroxidase (POD) and glutathione oxidase (GSHOx) mimicking activities, and photothermal conversion performance, which can result in ferroptosis featuring the up-regulation of lipid peroxides (LPO) and reactive oxygen species (ROS). The accumulation of LPO and ROS provides a powerful approach for cleaving HSPs, which enables Pd SAzyme-mediated mild-temperature PTT.

A Label-Free Fluorescent AND Logic Gate Aptasensor for Sensitive ATP Detection.

In this study, a label-free fluorescent, enzyme-free, simple, highly sensitive AND logic gate aptasensor was developed for the detection of adenosine triphosphate (ATP). Double-stranded deoxyribonucleic acid (DNA) with cohesive ends was attached to graphene oxide (GO) to form an aptasensor probe. ATP and single-stranded DNA were used as input signals. Fluorescence intensity of PicoGreen dye was used as an output signal. The biosensor-related performances, including the logic gate construction, reaction time, linearity, sensitivity, and specificity, were investigated and the results showed that an AND logic gate was successfully constructed. The ATP detection range was found to be 20 to 400 nM (R² = 0.9943) with limit of detection (LOD) of 142.6 pM, and the sensitivity range was 1.846 × 106 to 2.988 × 106 M-1. This method for the detection of ATP has the characteristics of being simple, low cost, and highly sensitive.

The RNA/DNA-binding protein PSF relocates to cell membrane and contributes cells' sensitivity to antitumor drug, doxorubicin.

Cell surface proteins play an important role in multidrug resistance (MDR). However, the identification involving chemoresistant features for cell surface proteins is a challenge. To identify potential cell membrane markers in hematologic cancer MDR, we used a cell- and antibody-based strategy of subtractive immunization coupled with cell surface comparative screening of leukemia cell lines from sensitive HL60 and resistant HL60/DOX cells. Fifty one antibodies that recognized the cell surface proteins expressed differently between the two cell lines were generated. One of them, the McAb-5D12 not only recognizes its antigen but also block its function. Comparative analysis of immunofluorescence, flow cytometry, and mass spectrum analysis validated that the membrane antigen of McAb-5D12 is a nucleoprotein-polypyrimidine tract binding protein associated splicing factor, PSF. Our results identified that PSF overexpressed on the membrane of sensitive cells compared with resistant cells and its relocation from the nuclear to the cell surface was common in hematological malignancy cell lines and marrow of leukemia patients. Furthermore, we found that cell surface PSF contributed to cell sensitivity by inhibiting cell proliferation. The results represent a novel and potentially useful biomarker for MDR prediction. The strategy enables the correlation of expression levels and functions of cell surface protein with some cell-drug response traits by using antibodies.

NIR-Activatable Heterostructured Nanoadjuvant CoP/NiCoP Executing Lactate Metabolism Interventions for Boosted Photocatalytic Hydrogen Therapy and Photoimmunotherapy.

Near-infrared (NIR) laser-induced photoimmunotherapy has aroused great interest due to its intrinsic noninvasiveness and spatiotemporal precision, while immune evasion evoked by lactic acid (LA) accumulation severely limits its clinical outcomes. Although several metabolic interventions have been devoted to ameliorate immunosuppression, intracellular residual LA still remains a potential energy source for oncocyte proliferation. Herein, an immunomodulatory nanoadjuvant based on a yolk-shell CoP/NiCoP (CNCP) heterostructure loaded with the monocarboxylate transporter 4 inhibitor fluvastatin sodium (Flu) is constructed to concurrently relieve immunosuppression and elicit robust antitumor immunity. Under NIR irradiation, CNCP heterojunctions exhibit superior photothermal performance and photocatalytic production of reactive oxygen species and hydrogen. The continuous heat then facilitates Flu release to restrain LA exudation from tumor cells, whereas cumulative LA can be depleted as a hole scavenger to improve photocatalytic efficiency. Subsequently, potentiated photocatalytic therapy can not only initiate systematic immunoreaction, but also provoke severe mitochondrial dysfunction and disrupt the energy supply for heat shock protein synthesis, in turn realizing mild photothermal therapy. Consequently, LA metabolic remodeling endows an intensive cascade treatment with an optimal safety profile to effectually suppress tumor proliferation and metastasis, which offers a new paradigm for the development of metabolism-regulated immunotherapy.

Rational Design of Platinum-Bismuth Sulfide Schottky Heterostructure for Sonocatalysis-Mediated Hydrogen Therapy.

Conventional sonodynamic therapy is unavoidably limited by the tumor microenvironment, although many sonosensitizers have been developed to improve them to a certain extent. Given this, a concept of sonocatalytic hydrogen evolution is proposed, which is defined as an oxygen-independent therapeutics. To demonstrate the feasibility of the concept, the narrow-bandgap semiconductor bismuth sulfide (Bi2 S3 ) is selected as the sonocatalyst and platinum (Pt) nanoparticles are grown in situ to optimize their catalytic performance. In this nanocatalytic system, the Pt nanoparticles help to capture sonoexcited electrons, whereas intratumoral overexpressed glutathione (GSH), as a natural hole sacrificial agent, can consume sonoexcited holes, which greatly improves the charge-separation efficiency and promotes controllable and sustainable H2  generation. Even under hypoxic conditions, the Pt-Bi2 S3  nanoparticles can also produce sufficient H2  under ultrasound irradiation. Mechanistically, mitochondrial dysfunction caused by H2  and intratumoral redox homeostasis destruction by GSH depletion synergistically damage DNA to induce tumor cells apoptosis. At the same time, the Pt nanoparticles and holes can also trigger the decomposition of hydrogen peroxide into O2  to relieve tumor hypoxia, thus being synergistic with GSH depletion to reverse tumor immunosuppressive microenvironment. The proposed sonocatalysis-mediated therapy will provide a new direction to realize facile and efficient cancer therapy.

Cultivation Model of Entrepreneurship From the Perspective of Artificial Intelligence Ethics.

Students are the backbone of national development and progress, and should stand at the forefront of the era of innovation and entrepreneurship. Cultivating the entrepreneurship of college students is not only a response to the national call, but also a basic requirement for implementing quality education and promoting the comprehensive development of college students. To better study the entrepreneurship of college students, cultivate a group of newcomers of the era who have patriotic feelings, dare to innovate, hardworking and sustainable struggle, and solve the problem of college students' employment difficulties from the root, the cultivation mode of college students' entrepreneurship is studied. Firstly, the impact of artificial intelligence (AI) technology on social ethics is analyzed. Secondly, it analyzes the current situation of the cultivation of science and engineering college students' entrepreneurship from three aspects: Chinese traditional cultural thoughts influence the concept of career choice, enterprises emphasize utilitarianism, and colleges and universities attach importance to knowledge education and despise spiritual education. Finally, the data statistics on the cultivation of entrepreneurship of science and engineering college students are carried out in the form of questionnaires. The results demonstrate that among the students surveyed, 21.31% have a strong willingness to start their own business, and 72.84% have the idea of starting their own business, which means that most students still want to start a business through their own efforts, not blindly looking for jobs. Simultaneously, among many majors, 87.5% of students majoring in agriculture and medicine are better at finding new ways to solve problems, while the proportion of students majoring in literature and history is the lowest. It also indicates that most students believe that schools should add more seminar courses, internship courses, design courses, experimental courses, etc., and allow students to choose learning courses across colleges and majors, to cultivate college students' entrepreneurship. The proposed method provides some ideas for the application of AI technology in the cultivation of students' entrepreneurship.

Runtime verification in uncertain environment based on probabilistic model learning.

Runtime verification (RV) is a lightweight approach to detecting temporal errors of system at runtime. It confines the verification on observed trajectory which avoids state explosion problem. To predict the future violation, some work proposed the predictive RV which uses the information from models or static analysis. But for software whose models and codes cannot be obtained, or systems running under uncertain environment, these predictive methods cannot take effect. Meanwhile, RV in general takes multi-valued logic as the specification languages, for example the true, false and inconclusive in three-valued semantics. They cannot give accurate quantitative description of correctness when inconclusive is encountered. We in this paper present a RV method which learns probabilistic model of system and environment from history traces and then generates probabilistic runtime monitor to quantitatively predict the satisfaction of temporal property at each runtime state. In this approach, Hidden Markov Model (HMM) is firstly learned and then transformed to Discrete Time Markov Chain (DTMC). To construct incremental monitor, the monitored LTL property is translated into Deterministic Rabin Automaton (DRA). The final probabilistic monitor is obtained by generating the product of DTMC and DRA, and computing the probabilities for each state. With such a method, one can give early warning once the probability of correctness is lower than a pre-defined threshold, and have the chance to do adjustment in advance. The method has been implemented and experimented on real UAS (Unmanned Aerial Vehicle) simulation platform.

A tumor microenvironment-responsive Co/ZIF-8/ICG/Pt nanoplatform for chemodynamic and enhanced photodynamic antitumor therapy.

Hypoxia and the overexpression of hydrogen peroxide (H2O2) in the tumor microenvironment (TME) are conducive to cancer cell proliferation, which greatly hinders cancer treatment. Here, we design a novel TME-responsive therapeutic nanoplatform Co/ZIF-8/ICG/Pt (CZIP) to achieve chemodynamic therapy (CDT) and enhanced photodynamic therapy (PDT). In this nanoplatform, under near-infrared light (NIR) irradiation, the photosensitizer indocyanine green (ICG) can generate singlet oxygen (1O2) for cancer cell apoptosis. Meanwhile, overexpressed H2O2 in the TME could be catalyzed to generate O2 by the loaded Pt to relieve tumor hypoxia and promote the PDT-induced 1O2 production. In addition, the doped Co2+ could react with H2O2 to produce hydroxyl radicals (˙OH) for CDT. The multifunctional nanoplatform CZIP showed high biosafety and a good antitumor effect, which would provide a new route for cancer therapy.

Anoikis induction and metastasis suppression by a new integrin αvß3 inhibitor in human melanoma cell line M21.

Integrin αvß3 plays a critical role in the survival and metastasis process of cancer cells. It is therefore desirable to develop new types of small molecule inhibitors of integrin αvß3. IH1062 (3, 5-dichloro-phenylbiguanide) is a novel small molecule inhibitor of integrin αvß3 that we have recently discovered. In this study, we investigated the induction effects of anoikis in human melanoma cell line M21 by IH1062, by detecting caspase activity, measuring the expression levels of apoptosis-related proteins, and performing the AnnexinV/PI apoptosis assay. Furthermore, we established a melanoma pulmonary metastasis mouse model in order to evaluate the suppression of metastasis by IH1062 in vivo. Our results demonstrate that IH1062 triggered human melanoma M21 cells to undergo anoikis by interrupting the attachment of M21 cells to extracellular matrix, reducing the phosphorylation of focal adhesion kinase, decreasing survivin and the ratio of Bcl-2/Bax proteins, and activating caspase cascades in vitro. Additionally, IH1062 showed markedly anti-metastatic effects in the pulmonary metastasis model in vivo, which makes it a promising lead to develop new drugs for anti-metastasis therapies.

Improvement of tumor targeting and antitumor activity by a disulphide bond stabilized diabody expressed in Escherichia coli.

We have generated an anti-Pgp/anti-CD3 diabody which can effectively inhibit the growth of multidrug-resistant human tumors. However, the two chains of the diabody are associated non-covalently and are therefore capable of dissociation. Cysteine residues were introduced into the V-domains to promote disulphide cross-linking of the dimer as secreted by Escherichia coli. Compared with the parent diabody, the ds-Diabody obtained was more stable in human serum at 37 degrees C, without loss of affinity or cytotoxicity activity in vitro. Furthermore, the ds-Diabody showed improved tumor localization and a twofold improved antitumor activity over the parent diabody in nude mice bearing Pgp-overexpressing K562/A02 xenografts. Our data demonstrate that ds-Diabody may be more useful in therapeutic applications than the parent diabody.

[Effect of calmodulin antagonist O-4-ethoxyl- butyl-berbamine on the proliferation of human breast cancer cell line MCF-7 and its relevant mechanism].

OBJECTIVE: To investigate the effect of calmodulin antagonist O-(4-ethoxyl-butyl)-berbamine (EBB) on proliferation of human breast cancer cell MCF-7 and its possible mechanism. METHODS: MTT assay was used to analyze the effect of EBB on tumor cells growth. Flow cytometry was used to detect its impact on the cell cycle of MCF-7 cells. Immunofluoresce labeling technique and laser scanning confocal microscope were used to reveal the changes of the microtubule, microfilament, mitochondrion, and endoplasmic reticulum in the cells. RESULTS: The IC50 value of EBB in MCF-7 cells was (13.0 +/- 3.7) micromol/L. MCF-7 cells were arrested at S phase after EBB treatment. Meanwhile, depolymerization of the microtubule and microfilament, impairment of the mitochondrion and swelling of endoplasmic reticulum were observed. CONCLUSION: EBB arrests MCF-7 cells at S phase by inhibiting the growth of MCF-7 cells, which may be related to the changes of structures and functions of the microtubule, microfilament, mitochondrion, and endoplasmic reticulum.

Extracellular domain of 4-1BBL enhanced the antitumoral efficacy of peripheral blood lymphocytes mediated by anti-CD3 x anti-Pgp bispecific diabody against human multidrug-resistant leukemia.

Our previous data have shown a significantly higher tumor response to anti-CD3/anti-Pgp bispecific diabody-mediated immunotherapy for P-glycoprotein (Pgp)-overexpressing K562/A02 cells, but a rapid tumor relapse occurred at 1 week after therapy. In an attempt to overcome tumor recurrence, we supplemented the previous therapy with extracellular domain of human 4-1BBL (ex4-1BBL) to regulate the activation of peripheral blood lymphocyte (PBL). As a result, this combination showed enhanced cytotoxicity in vitro and eradicated the multidrug-resistant xenografts of K562/A02 in nude mice. Furthermore, no tumor recurrence was observed within 100 days after the first treatment. Therefore, when used as an adjuvant, ex4-1BBL may improve the outcome of PBL-based immunotherapy.

Improvement in soluble expression levels of a diabody by exchanging expression vectors.

Accumulating evidence suggests that bispecific antibody fragments (BsAbs) seem set to join monoclonal antibodies as powerful therapeutic and diagnostic agents, particularly for targeting cancer. One type of recombinant BsAbs are diabodies, which are constructed from heterogeneous single-chain antibodies and can increase antigen-specific cytotoxicity in T cells by cross-linking tumor antigens with T cell associated antigens. Some diabodies, however, cannot be solubly expressed in sufficient quantities, which limits their use in clinical therapeutics. Previously we constructed an anti-CD20 x CD3 diabody, which effectively directs the lytic potential of cytolytic T cells toward CD20(+) malignant B cells and shows marked antitumor efficacy in vivo. Here, to increase the amount of soluble product for clinical trials, we used an alternative expression vector under the T7 promoter but retained the stII signal sequences to ensure that the expressed protein is secreted to the periplasm of Escherichia coli. We achieved a periplasmic, soluble product by optimizing the conditions for induction with a yield of 8-9mg/L after affinity chromatography purification. This is nearly a five times greater yield than obtained with the previous vector. The diabodies generated from this modified vector retain dual binding specificity for both CD20-positive and CD3-positive cell lines. Taken together, these results suggest that changing expression vectors may be an alternative strategy to accomplish high-level expression of active BsAb proteins from E. coli.

Co-expression of cytokeratin 8 and breast cancer resistant protein indicates a multifactorial drug-resistant phenotype in human breast cancer cell line.

AIMS: The aim was to determine whether increased CK8 and BCRP expression cooperatively contribute to multidrug resistance (MDR) in MCF-7/MX cells. Accumulating evidence suggests that the development and maintenance of cancer MDR involves complex multimodal mechanisms that interact concomitantly and complementarily. In this report, we observed elevated expression of cytokeratin 8 (CK8) in MCF-7/MX, a mitoxantrone (MX)-selected human breast tumor cell line with the MDR phenotype known as overexpression of breast cancer resistant protein (BCRP). MAIN METHODS: Gene transfection methods were used to express CK8 and BCRP in NIH3T3 fibroblasts, individually or in combination. KEY FINDINGS: Taken together, our present study suggests that CK8 together with BCRP may play significant roles in conferring the multifactorial MDR phenotype of MCF-7/MX cells, but may act independently via potentially different mechanisms. Although expressing either CK8 or BCRP alone was able to confer resistance to mitoxantrone, cells co-expressing both proteins demonstrated significantly increased drug resistance. Furthermore, RNAi knockdown of CK8 and BCRP, alone and in combination, in MCF-7/MX cells significantly attenuated their resistance to chemotherapeutic agents. Interestingly, in contrast to inhibition of BCRP expression via anti-BCRP shRNA vector transfection, reversal of mitoxantrone resistance by transfection with anti-CK8 shRNA was not accompanied by an increase in intracellular drug accumulation. SIGNIFICANCE: Combinational approaches that target multiple drug-resistance-related molecules/pathways in cancer cells may represent more efficacious strategies to overcome MDR.